Quantitative Hippocampal StructuralChanges Following ElectroconvulsiveSeizure Treatment in a Rat Model of

DepressionSUSANNE S. KAAE,1,2 FENGHUA CHEN,1,2* GREGERS WEGENER,2,3 TORSTEN M. MADSEN,4

AND JENS R. NYENGAARD1

1Stereology and EM Laboratory, Centre for Stochastic Geometry and Advanced Bioimaging,Aarhus University Hospital, DK-8000 Aarhus C, Denmark

2Centre for Psychiatric Research, Aarhus University Hospital, DK-8240 Risskov, Denmark3Unit for Drug Research and Development, School of Pharmacy (Pharmacology), North-West University,

Potchefstroom, South Africa4H. Lundbeck A/S, International Clinical Research, Dept. 317, Ottiliavej 7-9, DK-2500 Valby, Denmark

KEY WORDS stereology; neurogenesis; gliogenesis; Flinders rats; forced swim test

ABSTRACT Objective: The pathophysiology of depression and the effects of anti-depressant treatment are hypothesized to be related to hippocampal structuralchanges. This study aims to investigate the effect of electroconvulsive seizures onbehavior and hippocampal structure in a rat model of depression. Methods: FlindersSensitive Line (FSL) and Flinders Resistant Line (FRL) rats were treated daily for 10days with either electroconvulsive seizures or sham treatment. The behavior was eval-uated using the forced swim test. Design-based stereological methods were used toquantify the hippocampal volume and the numbers of neurons and glial cells in spe-cific hippocampal subregions. Results: The basal level of hippocampal volume and neu-ron number differed significantly between the two rat strains, and a trend toward theFSL strain having more glial cells was found. The structural differences foundbetween the sham-treated animals were counteracted by electroconvulsive seizure(ECS) treatment, which also normalized the behavior. ECS treatment increased thenumber of glial cells in hilus significantly in the FRL rats and with the same tendencyfor the FSL rats. Conclusion: Our results indicate that along with hippocampal neuro-genesis, gliogenesis may also be involved in the pathophysiology of depression and inthe effect of antidepressant treatment. The underlying mechanisms remain unknown,and further investigations are required to clarify whether the structural changes arenecessary to induce a therapeutic effect of antidepressant treatment or if they ratherrepresent an epiphenomenon. Synapse 00:000–000, 2012. VVC 2012 Wiley Periodicals, Inc.

INTRODUCTION

Structural changes within the hippocampus may beintimately linked to the pathophysiology of depressivedisorders and, conversely, the mechanisms underlyingthe therapeutic effect of antidepressant treatment(Duman, 2004; Pittenger and Duman, 2008; Samuelsand Hen, 2011).

Magnetic resonance imaging studies have showndecreased hippocampal volumes of depressed individ-uals (Campbell and Macqueen, 2004; Sheline et al.,2003; Videbech and Ravnkilde, 2004). This has beensupported by findings of moderate apoptosis and atro-phy of hippocampal neurons in depressed patients(Campbell and Macqueen, 2004; Lucassen et al.,2001). This suggests that reduced hippocampal neuro-

genesis contributes to the pathogenesis of depression(Banasr and Duman, 2007; Lucassen et al., 2010), butchanges in glial morphology and number are also pos-sibly involved (Rajkowska and Miguel-Hidalgo, 2007)as well as reductions of the neuropil and volume

Contract grant sponsors: Danish Research Council, Lundbeck Foundation,Mind Centre, Danish Council for Strategic Research, Danish Medical ResearchCouncil, Villum Foundation.

*Correspondence to: Fenghua Chen, Stereology and Electron MicroscopyLaboratory, Danish Neuroscience Centre, 3rd Floor, Building 10G, AarhusSygehus, Aarhus University Hospital, Noerrebrogade 44, DK-8000 Aarhus C,Denmark. E-mail: fenghua.chen@ki.au.dk

Received 22 September 2011; Accepted 24February 2012

DOI 10.1002/syn.21553

Published online 5 March 2012 in Wiley Online Library (wileyonlinelibrary.com).

VVC 2012 WILEY PERIODICALS, INC.

SYNAPSE 00:000–000 (2012)

changes of the extracellular fluid (Czeh and Lucassen,2007).

Regarding the mechanisms underlying antidepres-sant treatment, hippocampal volume has been foundto be increased after electroconvulsive therapy (ECT)in depressed patients (Nordanskog et al., 2010). More-over, few studies have shown an increased volume ofdentate gyrus (DG) and a higher number of dividingcells in pharmacologically treated patients (Boldriniet al., 2009). More animal studies have shown anincreased number of neurons in the hippocampus af-ter antidepressant treatment (Chen et al., 2009, 2010;Hellsten et al., 2002; Madsen et al., 2000; Malberget al., 2000; Sahay and Hen, 2007). Furthermore,other animal studies have indicated that a behavioraleffect of antidepressants is dependent on hippocampalneurogenesis (Santarelli et al., 2003). Conversely, theinhibitory effects of stress on neurogenesis have beenfound to be diminished by different antidepressanttreatments (Duman, 2004; Lucassen et al., 2010).However, in other animal models, the behavioraleffect of antidepressant treatment seemed not todepend on neurogenesis (Castren and Rantamaki,2010; Sahay and Hen, 2007). Along with neurogene-sis, gliogenesis has also been suggested as animportant factor contributing to the effects of antide-pressant treatment (Czeh and Lucassen, 2007).Experimental studies have suggested that antidepres-sant treatment can induce changes in gliogenesis,glial morphology, and glial cell numbers (Czeh et al.,2006). Also, synaptic plasticity has been proposed asa factor involved in the mechanisms underlying anti-depressant treatment. Animal studies have demon-strated changes in synapse types and numbers afterantidepressant treatment (Chen et al., 2008, 2009,2010; Hajszan et al., 2009, 2010).

To study the pathophysiology of depression and theeffects of treatment, we used the Flinders SensitiveLine (FSL) rats as a genetic animal model of depres-sion. FSL rats were originally bred to have high sen-sitivity to the anticholinesterase diisopropyl flouro-phosphate (DFP) and, thus, resemble depressedhumans with regard to cholinergic supersensitivity.Compared with the FSL rats, the Flinders ResistantLine (FRL) rats have a lower sensitivity to DFP andare generally used as the corresponding control line(Overstreet et al., 2005). The FSL rat model ofdepression fulfills the major criteria of high face, con-struct and predictive validities for research on thepathophysiology of depression (Overstreet, 1993;Overstreet et al., 2005; Wegener et al., 2012b).

Today, ECT, with the corresponding animal modelelectroconvulsive seizure (ECS), is the fastest actingand most efficient treatment of depression used in theclinic (Husain et al., 2004; UK_ECT_Review_Group,2003). However, the underlying mechanism of actionremains unknown. ECT does not induce remission in

all patients and some patients still suffer from sideeffects.

The aim of this study was therefore to investigatewhether repeated ECS induces structural changes inthe hippocampus of the FSL rats. We used design-based stereology to estimate the hippocampal volumeand numbers of neurons and glial cells within thesubregions of hippocampus. Furthermore, the behav-ioral response after treatment was measured by theforced swim test (FST).

MATERIALS AND METHODSAnimals

Adult male FSL rats (n 5 16, 8/ECS, 8/sham) andFRL rats (n 5 16, 8/ECS, 8/sham) from breeding colo-nies maintained in the animal quarters of Centre forPsychiatric Research, Aarhus University Hospitalwere used. The animals derived from the FSL colonymaintained at the Karolinska Institute, Stockholm,Sweden, and were kept on a normal 12 h light/12 hdark cycle and had free access to food and water. Allrats were placed in the same room with two rats percage. All rats were prehandled for 3 min per day for 7days before any procedure was initiated. The bodyweight of all animals was measured after the lasttreatment. The study protocol was approved by theDanish animal ethics committee (approval number2007/561-1378).

ECS treatment

Animals were treated once daily for 10 days, everyday at 9 A.M. ECS was given via ear clip electrodesusing 55–70 mA in 0.5 s at a frequency of 100-Hzsquare wave pulses (UgoBasile, Comerio, Italy). Theaim of the treatment was to achieve a full tonic-clonicseizure at every administration of current. A pilotexperiment revealed that the FRL rats reached thiscriterion at currents of 55 mA, whereas the FSL ratsrequired 70 mA. Importantly, following ECS, all ratswere monitored for seizures ensuring that clonic move-ments of the face and forelimbs (indicating generalizedseizures) lasted for at least 10 s. Sham treatment usedthe same procedure but with no current passed.

Porsolt FST

To evaluate depression-like behavior and the anti-depressant efficacy of ECS in the FSL/FRL model, weused the Porsolt FST (Porsolt et al., 1978). A modifiedversion of the original procedure was used (Wegeneret al., 2012a). Briefly, the swim test was conducted byplacing the rat in a vertical cylinder containing 258Cwarm water (size: height 3 diameter 5 60 cm 3 24 cmand water depth 5 40 cm). The water was deep enoughto prevent the rat’s tail from touching the bottomof the cylinder. On day 10, a 15-min pretest was

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performed to habituate the animals to the experimen-tal condition. On day 11, rats were placed in the cylin-der for 7 min. During this time, the behavior of therats was registered using a video camera, and the du-ration of immobility was measured in seconds duringthe first 5 min of the test. Immobility during the swimtest was defined as both front paws being immobile. Toregister immobility, an automatic tracking system,Noldus Ethovision XT, was used (Noldus BV, Waache-ningen, Holland). After the swim test, the rats wereremoved from the cylinder, dried with towels, andplaced in a warm enclosure before they were returnedto their home cages.

Tissue preparation

Perfusion fixation of the animals and dissection ofthe brain were done 15 h after the last ECS or shamtreatment. Phenobarbital sodium (Unikem A/S, Co-penhagen, Denmark) was used for anesthesia, andtranscardial perfusion was done using 4% paraformal-dehyde and 2% glutaraldehyd in 0.1 M phosphatebuffer. After dissection, the brains were kept in thesame fixative at 48C until further processing. All ratswere randomized between groups when to performPorsolt swim test, so all brain underwent same treat-ment and also after fixation.

For sectioning, the right or left hippocampus wasrandomly chosen. The hippocampus was stretchedalong the septotemporal axis to diminish its naturalcurvature. The straightened hippocampus was embed-ded in 5% agar and, subsequently, sections were cutcontinuously on a Vibratome 3000 (Vibratome, StLouis, MO). The cutting was performed perpendicularto the longest axis and the section thickness was 65lm. Every 15th section was sampled using the princi-ple of systematic random sampling, giving a sectionsampling fraction (SSF) 5 1/15. This provided eightto nine sections per hippocampus.

The sections were mounted on chrome-gelatin-coated slides and Nissl stained with a 0.25% thioninesolution (thionin, Sigma T3387) before further investi-gation in a light microscope.

Definition of anatomical areas

We investigated the hippocampal formation, includ-ing the granule cell layer (GCL), the hilus of the DG,and the subdivisions of the cornu ammonis (CA; Fig.1). The numbers of neurons were estimated in the fol-lowing anatomical divisions: (1) GCL of the DG,including also the subgranular zone, (2) the hilus ofthe DG, and the pyramidal cell layers of (3) CA2/3and (4) CA1, respectively. In the case of glial cells,the areas of interest were as follows: (1) the hilus ofthe DG, stratum radiatum (str. rad.) of (2) CA2/3 and(3) CA1, respectively, and (4) the stratum lacunosummoleculare (slm.) of the CA1.

The GCL of DG appeared as a horseshoe-shapedlayer, with the soma of the neurons being small insize and very densely packed. Between the blades ofthe GCL is the hilus, a polymorphic region mainlycomposed by loosely packed inhibitory mossy cells,the mossy fibers from the granule cells, and glialcells. In a Nissl-stained section of the hippocampus,the hilus appeared light because of the low number ofcell bodies. The midpoint of the inner border of the hi-lus abuts the medial end of the well-defined pyrami-dal layer of CA2/3.

In this study, the layers of the CA regions weredefined as follows: as the CA2 region is very smalland only differs from the CA3 in the way that the py-ramidal cells are not receiving any mossy fibers, theCA2 and CA3 in this study were combined in oneregion named the CA2/3. Pyramidal cell layers ofCA2/3 are easily distinguished from the stratum radi-atum internally placed and the stratum oriens towardthe outside. The border between the CA2/3 and CA1is defined by a small transition zone between the tworegions. The transition zone contains cells varying insize and being less densely packed. Compared withthe CA2/3, the size of the pyramidal cells and theirnucleoli is smaller in the CA1. The border of the CA1toward the subiculum is defined by the pyramidalcells ceasing to be contiguous (West et al., 1991). Weincluded the stratum lucidum of CA3 to the CA2/3stratum radiatum. The stratum radiatum of CA2/3and CA1, respectively, was defined by the locationinside the right adjacent pyramidal cell layers. Theinner border of the CA2/3 stratum radiatum wasdefined by a decrease in cell density, whereas theinner border of CA1 stratum radiatum was defined bythe stratum lacunosum moleculare, which has ahigher density of cells and a distinct course of thefibers.

Fig. 1. The rat hippocampal formation seen in light microscopeafter thionin staining. The investigated anatomical subregions aredelineated: granule cell layer of the dentate gyrus (DG), hilus, py-ramidal cell layer of CA2/3 and CA1 (CA2/3 and CA1), stratum radi-atum of CA2/3 and CA1 (CA2/3 str. rad. and CA1 str. rad.), andstratum lacunosum moleculare of CA1 (CA1 slm.)

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Stereological counting

The counting process was performed using the new-CAST system (Visiopharm, Hørsholm, Denmark).This system consists of an Olympus light microscope(Olympus BX50, Olympus, Denmark) modified forstereology with a digital camera (PixeLINKPLA686C, Canada) and a motorized microscope stage(Prior H138 with controller H29, Cambridge, UK).The newCAST software was interfaced to the digitalcamera superimposing the counting frames on thelive images.

Within each section, the specific areas of interestwere delineated as shown in Figure 1.

Estimating the number of neurons usingthe optical fractionator

The optical fractionator (Dorph-Petersen et al.,2001; Gundersen, 1986; West et al., 1991) with vary-ing sampling fractions was used to count neurons orglial cells disregarding their size, shape, and orienta-tion. All counting was performed by the same person,who was blinded to the group assignment of the case.

Within each of the subregions, systematic randomsampling was performed at high magnification usinga known area sampling fraction (ASF) appropriate forthe specific subregion. Depending on which subregionwas investigated, the counting was done using either603 (hilus, str. rad., and slm.) or 1003 (GCL and py-ramidal cell layer) oil immersion lenses. To count neu-rons, the nucleolus was used as counting unit, as neu-rons have one nucleolus only. To count glial cells, thewhole cell soma was used as counting unit. As endo-thelial cells might be mistaken for glial cells, all cellsplaced right next to a vessel could not be counted.Glial cells can be distinguished from neurons basedon a thionin staining (Nissl stain) according to theirsmaller size and lack of a nucleolus and stained cyto-plasm. Most glial cells can easily be distinguished byvisual inspection of their morphology. Oligodendro-cytes display more compact, darkly stained nucleieccentrically located in one end of a polar cell body.Astrocytes have larger and lightly stained nuclei andlack of stained cytoplasm. Microglia are characterizedby irregular shape, darkly stained nuclei and patchesof condensed chromatin with little cytoplasmic stain-ing (Fitting et al., 2008, 2010; Fig. 2). However, weknow that microglia are not discernable with a Nisslstain, while oligodendroglia appear as small darknuclei with dark chromatin. Hamidi et al. showedthat it is difficult to reliably distinguish betweenastrocytes and microglia with a Nissl stain (Hamidiet al., 2004). Therefore, we combined all glial cells inour counting.

There is some tissue deformation because of thepreparation, and cells may partly be lost along thecut surface. To avoid bias caused by these factors, a

Z-axis analysis was performed, and disector countingwas done only in a fraction of the whole height of thesection. The disector was placed 15 lm under the topplane and continued for 10 lm in the depth of the tis-sue where the Z-axis distribution found the cell den-sity to be constant (in the depth from 15 to 25 lm).As there is a homogenous nonuniform shrinkage inthe Z-axis of thick vibratome sections, a weightedestimate of the section thickness was calculated basedon the number of units counted in that field of view(Dorph-Petersen et al., 2001). The height of the disec-tor and the mean of the weighted height of the sec-tion provided a height sampling fraction (HSF). Thecounting was done by focusing up and down throughthe tissue, and a neuron was counted when the nucle-olus came into focus inside the unbiased countingframe without touching the forbidden lines of theframe.

At last, an unbiased estimate of the total number,N, of neurons or glial cells in a specific subregion wascalculated from the number of counted units, Q2, andthe known sampling fractions, HSF, ASF, and SSF(explained earlier): N ¼ 1

HSF � 1ASF � 1

SSF �P

Q�:

Estimating the volume using thecavalieri principle

The Cavalieri principle (Gundersen and Jensen,1987) was used for quantifying the total volume ofthe hippocampus, including all layers of the DG andCA subdivisions, using a 43 lens. The average areashrinkage in the X–Y-direction of these vibratome

Fig. 2. The image from the Nissl-stained section includes neu-rons and different subtypes of glial cells. N, neuron (note the nucle-olus); A, astrocyte (a larger, irregularly shaped and lightly stainedastrocyte nucleus); O, oligodendrocyte (more compact, darklystained nuclei eccentrically located in one end of a polar cell body);M, microglia cell (small irregular shape and darkly stained nuclei).Because of the large thickness of these sections one image of onefocal plane does not give justice to the ability to identify these cellsdue to ‘‘out-of-focus information.’’ [Color figure can be viewed in theonline issue, which is available at wileyonlinelibrary.com.]

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sections is 1–2%, a procedure that is checked regu-larly in our laboratory. The microtome advance of ourvibratome is also checked on a regular basis andvaries less than 1% when set on 65 lm. Thus, the vol-ume estimate is basically unaffected by tissue shrink-age (Dorph-Petersen et al., 2001).

Statistics

For statistical analysis, Statistical Package for theSocial Sciences (SPSS Corp, Chicago, IL) programwas used. Graphical presentations were made usingSigmaplot 10 (SYSTAT, San Jose, CA).

To compare treatment responses, all data were sub-jected to two-way analysis of variance (ANOVA) withstrain and treatment as the selected factors. If theANOVA revealed significant differences betweengroups, a post hoc test [Fisher least significant differ-ence (LSD) test] was performed to elucidate any pat-tern of group differences.

Furthermore, analysis of covariance (ANCOVA) wasconducted regarding the influence of the body weight onthe relation between hippocampal volume and strain.

Coefficient of error (CE) was evaluated according toGundersen et al. (1999). A P-value less than 0.05 wasconsidered statistically significant.

RESULTSDescriptive data

The body weight was not significantly influenced byeither strain or treatment, but there was a significantstrain 3 treatment interaction effect (F3, 28 5 7.24,P < 0.05; Table I). The FSL sham group had signifi-cantly lower body weight compared with the FRLsham group (P < 0.01).

The age was not influenced by either strain ortreatment, and there was no strain 3 treatmentinteraction effect.

We compared the volume of hippocampus by‘‘strain’’ with ‘‘weight’’ as covariates. We ran theANCOVA with the software SPSS in which the vol-ume of hippocampus is the dependent variable, rat isthe independent variable, and weight is the covariate.The results showed that there was no significantinteraction with ‘‘strain’’ and ‘‘weight’’ (F3, 28 5 3.38;P > 0.05).

Forced swim test

In the FST, the immobility was significantly influ-enced by strain (F3, 28 5 174.33; P < 0.001) and treat-ment (F3, 28 5 188.88; P < 0.001) with a significantstrain 3 treatment interaction effect (F3, 28 5 97.34; P< 0.001). The FSL sham group displayed significantlyhigher immobility in the FST compared with the FRLsham group (P < 0.001). Following treatment, the FSLECS group showed a significant decrease in immobilitycompared with the FSL sham group (P < 0.001), thisalso being true for the FRL ECS group compared withthe FRL sham group (P < 0.05; Fig. 3A; Table II).

Hippocampal volume

The volume of the hippocampus was significantlyinfluenced by strain (F3, 28 5 19.6; P < 0.05) andtreatment (F3, 28 5 65.34; P < 0.001) without a signifi-cant strain 3 treatment interaction. The resultsshowed that the volume of the hippocampus in theFRL sham group was significantly larger than that ofthe FSL sham group (P < 0.05). The volume of thehippocampus in the FSL ECS group and the FRL ECSgroup was significantly increased after ECS treatmentcompared with the FSL sham group and FRL shamgroup, respectively (P < 0.001; Fig. 3B; Table II).

Number of neurons

The number of neurons in the DG was significantlyinfluenced by treatment (F3, 28 5 8,50; P < 0.05) andalso seemed to be influenced by strain (F3, 285 2.98; P50.096) and strain3 treatment (F3, 285 4.02; P5 0.055).

The number of neurons in the DG of hippocampuswas significantly lower in the FSL sham group com-pared with the FRL sham group (P < 0.05). No differ-ences in neuron number were found between thesetwo groups in the hilus, CA1, and CA2/3 subregions.There was, however, a tendency that the FRL shamgroup had more neurons in the CA1 and CA2/3 subre-gions than did the FSL sham group.

In the FSL group, repeated ECS treatment resultedin a significant increase in the number of neurons inthe dentate GCL when compared with the sham-treated FSL group (P < 0.01). No similar changewas found in the FRL group after ECS treatment(Fig. 3C; Table II).

TABLE I. Descriptive data

FRL sham FRL ECS FRL sham vs. FRL ECS FRL sham vs. FSL sham

Mean (SD) Mean (SD) P P

Body weight (g) 275.6 (45.5) 230.4 (48.1) 0.051 0.003**

Age (days) 64.5 (9.7) 62.5 (10.5) – –FSL sham FSL ECS FSL sham vs. FSL ECSMean (SD) Mean (SD) P

Body weight (g) 203.0 (50.0) 242.3 (31.7) 0.088Age (days) 62.0 (6.3) 65.3 (8.1) –

P-values in post hoc Fishers LSD test,**<0.01.

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The number of neurons in the hilus was only signifi-cantly influenced by treatment (F3, 28 5 8.43; P <0.01) and not by strain. In the FRL-ECS group, thenumber of hilus neurons was significantly increasedcompared with the FRL sham group (P < 0.05). Nosignificant differences were found in the neuronalnumber between those two groups in the DG, CA1,and CA2/3 subregions. There was a tendency that theFSL-ECS group had more neurons in the hilus thandid the FSL sham group (P 5 0.094; Fig. 3D; Table II).

No changes were found in the CA1 or CA2/3 afterECS in either the FSL or the FRL group.

Number of glial cells

The number of glial cells in the hilus was signifi-cantly influenced by treatment (F3, 28 5 11.25; P <0.01), and it also seemed to be influenced by strain(F3, 28 5 3.57; P 5 0.069). The number of glial cells inthe hilus was significantly increased in the FRL ECS

Fig. 3. Graphs showing the statistical significant findings (P-value in post hoc Fishers LSDtest). (A) Duration of immobility in the forced swim test, (B) hippocampal volume, (C) numberof neurons in GCL of the DG, (D) number of neurons in the hilus, and (E) number of glial cellsin the hilus. *<0.05, **<0.01, and *** <0.001.

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group compared with the FRL sham group (P < 0.01),but no changes in glial number were found in theCA1 and CA2/3 subregions (Fig. 3E; Table II).

In the FRL sham group, there was a tendency forthe number of glial cells in the hilus being lower com-pared with the FSL sham group (P 5 0.052; Fig. 3E).However, no differences in glial numbers were foundin the CA1 and CA2/3 subregions.

DISCUSSION

In this study, we found the baseline levels of hippo-campal volume and neuron number in the GCL of theDG of the ‘‘depressed’’ FSL rats to be significantlylower compared with the FRL rats. Repeated ECSseemed to increase these parameters. For the firsttime in research, we demonstrated a tendency of thenumber of glial cells in the hilus to be influenced bystrain. Also, among FRL rats we found hippocampalvolume and number of neurons and glial cells in hilusto be increased after repeated ECS. Our study sup-ports the theory that specific structural changes inthe hippocampus are involved in the clinical responsefollowing ECS.

We used the FSL model to represent a depressedphenotype compared with the nondepressive pheno-type FRL. The phenotypic difference was demon-

strated by results of the FST, which found the FSLsham rats to be significantly more immobile than theFRL sham rats. This finding is consistent with previ-ous investigations of the same model (Overstreet et al.,2005). All rats used were male and had the same age.The body weight of the animals was measured as theFSL strain, in general, is known to weigh less than theFRL strain (Overstreet, 1993). Consistently, we foundthe FSL sham group to have significantly lower bodyweight compared with the FRL sham group.

The immediate effect of ECS was evaluated bymonitoring the duration of a clonic seizure. This wasdone to ensure that the rats received a treatmentcomparable to that given in human ECT. Importantly,not all rats needed the same current to achieve thetonic clonic seizures; thus, an interval from 55 to 70mA was used. This resembled the principle of humanECT, which aims to achieve the approximately sameduration of a seizure, but uses different currentsbetween patients. In general, the FSL rats needed ahigher current than the FRL rats, but the explana-tion for this and the impact on the results remainsunknown.

The hippocampal volume was significantly loweramong the FSL sham compared with the FRL shamrats. This finding is in line with findings done instudies of depressed patients (Campbell and

TABLE II. Results for all four groups of animals

FRL sham FRL ECSFRL sham

vs. FRL ECSFRL sham

vs. FSL sham

Mean (SD) OCV CE Mean (SD) OCV CE P P

Immobility (%) 17.38 (8.83) 0.508 – 7.500 (4.87) 0.649 – 0.011* <0.001***

Total volume (mm3) 32.60 (0.86) 0.026 – 37.33 (2.06) 0.055 – <0.001*** 0.043*

Total number of neuronsGranule cell in dentate gyrus (3105) 9.699 (2.47) 0.255 0.072 10.21 (0.83) 0.081 0.071 0.525 0.013*

Hilus of dentate gyrus (3104) 4.015 (0.88) 0.219 0.107 5.100 (0.70) 0.137 0.093 0.025* 0.463CA2/3 pyramidal layer (3105) 4.148 (1.04) 0.251 0.062 3.974 (1.02) 0.256 0.061 0.728 0.668CA1 pyramidal layer (3105) 3.233 (0.55) 0.169 0.061 3.269 (0.85) 0.260 0.061 0.962 0.554

Total number of glial cellsHilus of dentate gyrus (3105) 0.996 (0.09) 0.091 0.084 1.226 (0.15) 0.126 0.074 0.005** 0.052CA2/3 stratum radiatum (3105) 1.845 (0.37) 0.199 0.080 2.020 (0.43) 0.212 0.074 0.376 0.193CA1 stratum radiatum (3105) 1.196 (0.21) 0.173 0.078 1.231 (0.18) 0.146 0.076 0.720 0.897CA1 stratum lacunosum

moleculare (3105)1.190 (0.19) 0.157 0.098 1.234 (0.14) 0.111 0.095 0.756 0.177

FSL sham FSL ECSFSL sham

vs. FSL ECS

Mean (SD) OCV CE Mean (SD) OCV CE P

Immobility (%) 76.13 (7.57) 0.099 – 16.00 (6.97) 0.436 – <0.001***

Total volume (mm3) 30.73 (1.48) 0.048 – 36.07 (2.30) 0.064 – <0.001***

Total number of neuronsGranule cell in dentate gyrus (3105) 7.587 (1.20) 0.158 0.082 10.37 (1.43) 0.138 0.070 0.002**

Hilus of dentate gyrus (3104) 4.357 (0.84) 0.193 0.108 5.151 (1.18) 0.228 0.096 0.094CA2/3 pyramidal layer (3105) 3.938 (0.69) 0.175 0.062 4.062 (0.72) 0.176 0.061 0.782CA1 pyramidal layer (3105) 3.033 (0.70) 0.231 0.060 2.907 (0.79) 0.271 0.062 0.731

Total number of glial cellsHilus of dentate gyrus (3105) 1.149 (0.18) 0.153 0.079 1.275 (0.17) 0.130 0.074 0.104CA2/3 stratum radiatum (3105) 2.104 (0.34) 0.163 0.075 2.042 (0.41) 0.200 0.074 0.753CA1 stratum radiatum (3105) 1.183 (0.23) 0.192 0.078 1.196 (0.15) 0.128 0.076 0.898CA1 stratum lacunosum

moleculare (3105)1.385 (0.39) 0.280 0.091 1.380 (0.34) 0.248 0.090 0.968

P-values in post hoc Fishers LSD test,*<0.05,**<0.01, and***<0.001.

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Macqueen, 2004; Sheline et al., 2003; Videbech andRavnkilde, 2004). Our findings suggest that this vol-ume difference was, at least partly, due to a lowernumber of neurons in the GCL of DG. In the otherinvestigated subregions, only tendencies of fewer neu-rons were found. However, it could also be consideredthat a lower hippocampal volume might be correlatedto the lower body weight of the FSL sham group. Fur-thermore, the lower neuron number among FSL ratswas not necessarily due to disturbed neurogenesis butmight just reflect the lower volume. This might indi-cate that the neuronal density could still be the samein the two strains. However, lowered cell proliferationin the GCL of the FSL rats has previously been foundin a study by Bjornebekk et al. (2005). In contrast,studies by Petersen et al. found significantlyincreased cell proliferation in the hippocampus ofFSL rats compared with FRL rats (Petersen et al.,2008, 2009). However, the outcome may have beenaffected by the fact that they only investigated thedorsal part of the hippocampus.

As for the hippocampal neuron number in the FSLand FRL rats, respectively, interestingly, in one of ourprevious studies, using the same FSL/FRL model, sig-nificant differences in neuron numbers were indeedobserved in other subregions, hilus of the DG and py-ramidal cell layer of the CA2/3 and CA1 (Chen et al.,2010). No clear explanation for the discrepancybetween the present and our previous study exists.One possible explanation could be that the animals inthe present study derived from another line than thatused in the study by Chen et al. (2010).

As far as known to the authors, no previous studyhas investigated the hippocampal glial cells in theFlinders rat model. Thus, this study provides newknowledge about this rat depression model. So far,postmortem analyses of depressed patients havefailed to demonstrate major reductions in glial cellnumber in the hippocampus (Czeh and Lucassen,2007). However, reduced numbers have been found inother brain areas such as amygdala (Bowley et al.,2002) and prefrontal cortex (Ongur et al., 1998). Inthe current study, the number of glial cells in the hi-lus was almost significantly influenced by strain, withthe FSL strain having more glial cells. This is consist-ent with findings of increased glial packing density inall hippocampal subfields and DG of depressedhumans (Stockmeier et al., 2004) and supports thehypothesis of glial cells having a substantial role inthe pathogenesis of depression. A higher glial cellnumber in the hilus among the FSL sham group,although not significant, could be explained by moretheories. As both astroglia and microglia are knownto proliferate and become activated in response toneuronal injury (Rajkowska and Miguel-Hidalgo,2007), a higher glial cell number may reflect glial pro-liferation in response to the lowered neuron number

within the GCL of the DG. Another explanationmight be that the present glial cells are in some waydysfunctional, as a lack of neurotrophic factors from,e.g., glial sources is associated with higher vulnerabil-ity to cell damage among neurons (Rajkowska andMiguel-Hidalgo, 2007).

Comparing the sham-treated and ECS-treated ani-mals, we found that repeated ECS significantlyincreased the total hippocampal volume in bothstrains. This is consistent with the findings done in ahuman study (Nordanskog et al., 2010). Our findingssuggest that these volume changes were, at leastpartly, due to increased numbers of neurons and glialcells in specific hippocampal subregions. Further-more, our findings indicate that the structuralchanges induced by ECS were related to improvedbehavior among FSL rats. Other antidepressanttreatments have also been found to improve thebehavior among FSL rats in the FST (Chen et al.,2010; Overstreet et al., 2005; Petersen et al., 2009).In our study, the duration of immobility in the FSLECS group was decreased to the level of the FRLsham rats, meaning that repeated ECS was able todiminish the depressive behavior of the FSL rats. Wealso found reduced immobility among FRL rats afterrepeated ECS. Corresponding to our study, one studyof chronic ECS treatment (Jimenez-Vasquez et al.,2007) and one study of chronic administration of desi-mipramine (Overstreet et al., 2005) have also foundreduced immobility in FRL rats after treatment.

In the current study, the number of neurons wassignificantly increased in the GCL of DG afterrepeated ECS treatment among the FSL rats. Nochanges in other subregions were found, except for atendency of increased number of neurons in the hilus.These findings are consistent with the results fromthe study of imipramine in FSL rats (Chen et al.,2010) and agree with studies of other animal modelsfinding that ECS treatment increases hippocampalneurogenesis in the DG (Chen et al., 2009; Hellstenet al., 2002; Madsen et al., 2000). The present studydid not find any difference in neuron number in theGCL of DG between FRL sham and FRL ECS, but asignificant increase within the hilus was found. Pre-suming that the hippocampal neuron number influen-ces behavior, the increased number found among FRLrats might indicate one of the reasons why ECS treat-ment, compared with pharmacological treatments, ismore efficient in improving behavior. In the study ofimipramine (Chen et al., 2010) and in a study of theeffect of running (Bjornebekk et al., 2005), no changesin cell proliferation in FRL rats were found at all.

The increased neuron number after ECS amongboth FSL and FRL rats may have been due to eitheran increased rate of neurogenesis or a lower incidenceof neuronal death. However, it still remains unknownif the structural changes occurred before the behav-

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ioral changes or which mechanisms caused the struc-tural changes. More recent findings in the studies ofdifferent mouse strains are challenging the hypothesisthat increased hippocampal neurogenesis is necessaryto the behavioral effect of antidepressant treatment(Castren and Rantamaki, 2010; Sahay and Hen, 2007).Regarding the FRL/FSL model, a study by Petersenet al. showed that the behavioral improvementinduced by escitalopram was associated withdecreased hippocampal neurogenesis in maternallyseparated FSL rats (Petersen et al., 2008). In another,it was found that nortriptyline mediated behavioralimprovement in the FSL rats without affecting hippo-campal neurogenesis (Petersen et al., 2009). Both stud-ies evaluated neurogenesis in the DG but only in thedorsal part of hippocampus. The fact that we investi-gated the whole length of the hippocampus may havecaused our different results. Along the septotemporalaxis within the hippocampus, more differences withregard to cell proliferation have been found (Sahayand Hen, 2007). Chronic mild stress has been foundonly to decrease the cell proliferation in the ventralDG and not in the dorsal DG (Jayatissa et al., 2006).Escitalopram (Jayatissa et al., 2006) and agalometine(Banasr et al., 2006) have been found only to inducecell proliferation in the ventral and not in the dorsalhippocampus.

Consistent with findings done in other animal mod-els of depression, we found that repeated ECS signifi-cantly influenced the number of glial cells in the hi-lus. Glial proliferation in the hippocampus after ECShas also been found in other animal models (Wenn-strom et al., 2003, 2006). Furthermore, morphologicalchanges and activation of hippocampal glial cellshave been found in relation to ECS (Jansson et al.,2009). In the present study, the glial increase afterECS was only significant among FRL rats, whichmight be explained by a bigger brain and thus moreprogenitor cells. Our study suggests that the hilus ofthe DG seems to be of certain interest when investi-gating the role of glial cells in depression. Because ofthe methods used, we still do not know the subtypesof the glial cells involved. To understand the exactrole of the glial cells, more studies focusing on specificglial subtypes are needed.

Our study demonstrated that the volume and neu-ron number in the hippocampus were significantlysmaller in the ‘‘depressed’’ FSL rats. Furthermore, wefound that ECS induced an increase in the hippocam-pal volume and neuron and glial cell numbers alongwith a behavioral improvement. Thus, this study sup-ports the theory that both neuron and glial cell num-bers in the hippocampus have a role in the patho-physiology of depression and that the clinical effect ofECS treatment is due to counteraction of these struc-tural alterations. The underlying mechanism linkinghippocampal structural changes to the pathophysiol-

ogy of depression, however, continues to be unclear.Thus, further investigation is needed to decidewhether neurogenesis and gliogenesis are necessaryto induce a therapeutic effect of antidepressant treat-ment or if they simply represent an epiphenomenonor are rather related to the development of sideeffects.

ACKNOWLEDGMENTS

The authors thank Nadia G. Knudsen, Julie D.Jensen, and Heidi Jungland for their help in caringfor and overseeing the experimental animals. Wethank David H Overstreet for providing us with theinitial FSL/FRL breeding pairs. We also thankAleksander A. Mathe, Karolinska Institute, Karolin-ska University Hospital, Stockholm, Sweden, for pro-viding them with the initial FSL/FRL breeding pairs.

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