Cassidy Et Al., 2010_ Reelin PFC

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DEVELOPMENTAL EMERGENCE OF REELIN DEFICITS IN THEPREFRONTAL CORTEX OF WISTAR RATS REARED IN SOCIALISOLATION

A. W. CASSIDY, a S. K. MULVANY, a M. N. PANGALOS, b

K. J. MURPHY a AND C. M. REGAN a *a The Applied Neurotherapeutics Research Group, School of Biomo-lecular and Biomedical Science, UCD Conway Institute, University College Dublin, Beleld, Dublin 4, Ireland b Discovery Neuroscience, Wyeth Research, Princeton, NJ 08543,USA

AbstractAs the pathophysiological mechanism(s) of manyneuropsychiatric disorders relate to GABAergic interneuronstructure and function, we employed isolation rearing of Wistar rats as a model to correlate developmental emergenceof cognitive decits with the expression of reelin-producinginterneurons in the medial prefrontal cortex (PFC). Prepulseinhibition decits emerged at postnatal day 60 and persistedinto adulthood. Paralleling the emergence of these neurobe-havioural decits was an increase in reelin production andreelin-immunopositive cells in layer I of the PFC and this later became signicantly reduced at postnatal day 80. Cells ex-pressing reelin immunoreactivity in a horizontal orientationwere mainly located to the upper regions of layer I whereasthose with a vertical orientation, whose arbors extend intocortical layers II and III, were more numerous in the lower regions of layer I and became signicantly dysregulated dur-ing postnatal development. No behavioural decits or alteredreelin expression was observed at postnatal days 30 or 40.Developmental emergence of neurobehavioural and reelindecits in isolation reared animals is proposed to reectmaladaptive wiring within the medial prefrontal cortex duringa critical maturation period of this circuitry. 2010 IBRO.Published by Elsevier Ltd. All rights reserved.

Key words: prepulse inhibition, GABAergic interneurons, iso-lation rearing, layer I, immunouorescence, prelimbic cortex.

Studies of postmortem brain tissue have provided signi-

cant evidence that the GABAergic system is signicantlydisrupted in a number of neuropsychiatric disorders ( Lewiset al., 2005; Lisman et al., 2008 ). Such studies have showna reduction in protein markers of interneuron function inpost-mortem tissue obtained from the prefrontal cortex( Akbarian et al., 1995; Beasley and Reynolds, 1997; Sakaiet al., 2008 ), cingulate cortex ( Benes et al., 1991; Woo etal., 2004; Oblak et al., 2009 ), and temporal lobe ( Chanceet al., 2005 ). The reproducibility of these ndings supports

the possibility that interneuron decits are a central featurein the underlying pathophysiology of these disorders(Benes and Berretta, 2001; Fountoulakis et al., 2008;Lewis and Gonzlez-Burgos, 2008 ).

One subclass of GABAergic interneurons express andsecrete reelin ( Pesold et al., 1998, 1999 ), a large extracel-lular matrix glycoprotein ( 400 kDa) that has a wide arrayof functions in both the developing and adult cortex. Duringearly development reelin is synthesised by CajalRetziusinterneurons and plays a vital role in the lamination of cortical cell layers ( Tissir and Gofnet, 2003 ). In adulthoodreelin becomes more widely expressed by GABA interneu-rons, where it is believed to play a role in the renement of dendritic arbor and synapse formation ( Borrel et al., 1999;Costa et al., 2001; Dong et al., 2003; Boqoch and Linial,2008 ). Reelin contains eight repeats of 300350 aminoacids and, upon secretion into the extracellular space, iscleaved by metalloproteinases between repeats 23 and67, the central 36 repeats being required for activationof its receptor complex that is formed by the very low

density lipoprotein receptor and apolipoprotein E receptor 2 (Lambert de Rouvroit et al., 1999; Trommsdorff et al.,1999; Ignatova et al., 2004; Jossin et al., 2004 ).

The secretion of reelin into the extracellular spacesurrounding dendrites, dendritic spines and axon boutons(DArcangelo et al., 1995; Alcntara et al., 1998; Pappas etal., 2002; Tissir and Gofnet, 2003 ) is known to regulatesynapse structure and stability ( Weeber et al., 2002; Donget al., 2003 ). Evidence exists to implicate reelin decits inthe developmental emergence of a number of neuropsy-chiatric conditions including schizophrenia, bipolar disor-der (Torrey et al., 2005 ) and autism ( Fatemi et al., 2005; Ashley-Koch et al., 2007 ). For example in schizophrenia,reelin-mediated synapse plasticity appears to be compro-mised as both reelin mRNA and GAD67 mRNA expressionhave been found to be signicantly depressed in GABAergicinterneurons in the supercial layers of the prefrontalcortex ( Impagnatiello et al., 1998; Fatemi et al., 2000;Guidotti et al., 2000; Grayson et al., 2005; Torrey et al.,2005 ). These observations suggest altered reelin activitymay, in part, be responsible for the emergence of this andother psychotic states, however, postmortem tissue de-rived from patients at the end-stage of schizophrenia can-not provide an insight into the preceding developmentalmechanisms.

As many psychiatric disorders emerge during late ad-olescence ( Paus et al., 2008 ), animal models that recapit-

ulate their cardinal features in adulthood become a priorityin any attempt to understand the developmental mecha-

*Corresponding author. Tel: 353-1-716-6775; fax: 353-1-716-6920.E-mail address: [email protected] (C. M. Regan). Abbreviations: ANOVA, analysis of variance; BSA, bovine serum al-bumin; EDTA, ethylenediamine tetra acetic acid; HEPES, 4-(2-hy-droxyethyl)-1-piperazineethanesulfonic acid; NGS, normal goat se-

rum; PBS, phosphate-buffered saline; PFC, prelimbic region of pre-frontal cortex; PPI, prepulse inhibition; TBS-T, tris-buffered saline.

Neuroscience 166 (2010) 377385

0306-4522/10 $ - see front matter 2010 IBRO. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.neuroscience.2009.12.045

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nisms that lead to the onset of psychosis. Several modelshave been extensively studied and include repetitive ad-ministrations of NMDA antagonists ( Braun et al., 2007 ),neonatal ventral hippocampal lesion ( Lipska et al., 1993;Tseng et al., 2008 ) and rearing in isolation from time of weaning ( Geyer et al., 1993; Fone and Porkess, 2008 ). Ineach model, the behavioural phenotype includes hyperlo-comotion and prepulse inhibition decits. Most of theseabnormalities emerge during adolescence and in manycases there is also evidence of a reduction in markers of GABAergic interneurons ( Lipska et al., 2003; Pillai-Nair etal., 2005; Penschuck et al., 2006; Endo et al., 2007 ).

As reelin is implicated in the developmental structuring of the prefrontal cortex, a structure intimately involved in behav-ioural responses, we have correlated alteration of reelin pro-tein expression and reelin-secreting cells with emergence of behavioural decits in isolation reared animals.

EXPERIMENTAL PROCEDURES

Animal maintenance

Experimentally naive male Wistar rats were employed in all stud-ies. The animals were purpose bred at the Biomedical Facility,University College Dublin, and maintained in standard laboratoryconditions until the time of experimental use. Animals were intro-duced to the experimental holding rooms 5 days prior to thecommencement of the study, housed in groups of 34 during thisperiod, and maintained at 2224 C on a standard 12 h light/darkcycle, with food and water available ad libitum . On each of the 2days preceding commencement of behavioural studies, the ani-mals were handled and weighed and assessed in an open-eldarena for locomotor activity, rearing and general behaviour over a5 min period. All observations were carried out in the quiet roomunder low-level, red light illumination between 8:00 and 12:00 h tominimise the inuence of circadian rhythms. Isolation-reared ani-mals (isolated animals) were housed individually in non-soft bot-tom cages (225 345 170 mm), from time of weaning (postnatalday 25) until completion of behavioural testing. The standard 12 hlight/dark cycle was maintained and food and water was providedad libitum . Noise and visual stimuli were kept to an absoluteminimum as previously described ( Geyer et al., 1993 ). All exper-imental procedures were approved by the Animal Research EthicsCommittee of University College Dublin, conformed to EU CouncilDirective 86609-EEC, and were carried out by individuals retain-ing the appropriate licence issued by the Irish Department of Health. Throughout the course of these studies, every attemptwas made to ensure that the number of animals and any physical

distress was kept to an absolute minimum.

Sensorimotor gating

Pre-pulse inhibition served as an operational measure of senso-rimotor gating decits ( Swerdlow et al., 1994 ) and the protocolemployed was based on a procedure previously described byGeyer and colleagues (1993) . Each rat was restrained in anappropriately sized cylindrical holder, placed on a movement-sensitive platform and maintained in a soundproof chamber. Therat was allowed to habituate to a white noise background of 70 dBfor 5 min before receiving ve 20 ms startle trials of 120 dB,separated by randomised intervals of 1020 s. Immediately there-after, each rat received ve separate presentations with one of theprepulse stimuli of 72, 76, 80, or 84 dB and these were followed,100 ms later, by the 120 dB acoustic startle stimulus. Each trialwas separated by a time interval of 1020 s. The four prepulsestimuli were delivered in a randomised manner and included

periods in which there was no prepulse or startle stimulus. Thesession terminated with an additional ve startle trials. Signalswere integrated by the software supplied by the manufacturers of equipment hardware (MED-Associates Inc., St. Albans, VT, USA).

The effect of isolation rearing on pre-pulse inhibition was deter-mined in separate cohorts of animals ( n 78) on postnatal days30, 40, 60 and 80. The effects of isolation rearing on pre-pulseinhibition, as compared to that of the aged-matched cohorts(n 78) of social animals, were assessed by two-way analysis of variance (ANOVA) with post hoc analysis using a Bonferroni postt -test. In all cases, P -values less than 0.05 were considered to besignicant.

Immunoblot analysis of reelin expression

Tissue preparation. Separate cohorts ( n 4) of naive animalsreared in isolation or social groups were killed on postnatal days 30,40, 60 and 80, the brain removed and the medial prefrontal cortexdissected and excised. Samples were immediately placed in cryo-tubes, snap frozen in liquid nitrogen and stored in a 80 C freezer until required. Immediately prior to use, the samples were homoge-nised at 4 C in 300 l of 10 mM HEPES, pH 7.4, containing 0.32 Msucrose, 2 mM EDTA, and 0.01% of a protease and phosphataseinhibitor cocktail (Sigma, UK). The homogenates were subsequentlycentrifuged (1000 rpm, 15 min) and the supernatant was removedand stored at 20 C until further use.

SDS polyacrylamide gel electrophoresis and Western-blotting procedure. Protein concentrations were determined by theBCA assay, according to manufacturers instructions (Pierce,Rockford, IL, USA), and samples, of equal protein concentration,were boiled for 3 min in 70 mM TrisHCl, pH 6.8, containing 33mM NaCl, 1 mM EDTA, 2% (w/v) SDS, 0.01% (w/v) BromophenolBlue, 10% glycerol and 3% v/v dithiothreitol reducing agent. Thereduced and solubilised proteins were separated using pre-pre-pared 5% polyacrylamide gels and, subsequently, transferred tonitrocellulose membranes, according to manufacturers instruc-tions (Biorad, UK). Pre-stained molecular weight markers wereco-electrophoresed with the protein samples (Sigma, UK). Suc-cessful protein transfer was conrmed by staining the nitrocellu-lose sheet with Ponceau Red solution (Sigma, UK) prior to immu-noblotting and by Napthol Blue (Sigma, UK) following completionof immunoblotting. Reactive groups on the nitrocellulose sheetwere then inactivated using Tris buffered saline solution (TBS-T)blocking buffer (10 mM TrisHCl, pH 7.4, containing 150 mMNaCl, 0.05% (v/v) Tween-20, and with 5% (w/v) non-fat milkpowder) for 1 h at room temperature. The membrane was subse-quently incubated overnight (20 h) at 4 C in blocking buffer (5%v/v) containing a mouse monoclonal antibody to reelin (G10; Abcam, UK; 1:10,000 dilution). Following overnight incubation, thenitrocellulose membrane was washed three times in TBS-T pH 7.4

before being incubated for 1 h in blocking buffer containing ahorse radish peroxidase-conjugated anti-mouse IgG monoclonalantibody (Novagen, UK; 1:20,000 dilution). After incubation, thenitrocellulose sheet was washed three times in washing buffer,exposed for 5 min to a chemiluminescent peroxidase substrate(Pierce, Rockford, IL, USA) washed and exposed to X-ray lm(Fuji, UK) in a dark room under red light illumination, until optimalresolution of the protein bands was achieved. Immunoblot analy-sis of Reelin with the G10 antibody revealed three specic proteinbands, the full length 400 kDa protein and two bands at 300 and180 kDa, the latter representing the proteolytic fragments gener-ated by metalloproteinase cleavage at repeats 23 and 67,respectively. The X-ray lms were scanned, converted into adigital format and the immunostained band density analysed usingImageJ software ( http://rsb.info.nih.gov/ij/docs/index.html ). TheNaphthol Black-stained cellulose sheet was also scanned anddigitised and used to correct the immunostained bands for un-equal protein loading.

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Quantitative immunohistochemical analysis of reelinexpression in vivo

Tissue preparation. Animals were terminally anaesthetised

using 1 ml/kg sodium pentobarbital (Euthatal, Pzer AnimalHealth, UK) and their tissue was xed by transcardial perfusionwith a saline solution (0.9% w/v) for 2 min, followed by a 20 minperfusion with 0.12 M Srenson phosphate buffer (pH 7.2) con-taining 4% paraformaldehyde. The animals were then killed andtheir brains were removed and stored in 4% paraformaldehyde inSrensons buffer for a 24 h period. Following xation, the brainswere placed in a cryoprotective solution (40% sucrose [w/v] and10% glycerol [v/v] in dH 2 0) at 4 C for approximately 48 h. Thebrains were then coated in optimum cutting temperature com-pound (O.C.T.; Tissue-Tek, UK), to provide an even freezingrate, and lowered into a Cryoprep freezing apparatus (Algen Inc,UK) containing liquid CO 2 cooled n-hexane. The tissue was storedat 80 C until required.

Cryosectioning and immunohistochemical protocols. Sections

(16 m thick) of the prefrontal cortex were taken at level 3.2 mmrostral to bregma ( Paxinos and Watson, 1986 ) and placed in 0.32 Msucrose solution for 5 min. Free-oating sections were then washedtwice in 0.1 M (pH 7.4) PBS solution (phosphate buffered saline;Sigma, UK) solution for 10 min. Subsequently, the sections wereincubated in PBS containing 10% normal goat serum (NGS; Dako,DK) for 30 min, followed by incubation in a humidied chamber for 20 h with the G10 reelin monoclonal antibody diluted 1:1000 in PBScontaining 2% NGS and 2% bovine serum albumen (BSA; Sigma,UK). The sections were then washed twice in PBS for 10 min andfurther incubated for 3 h in the humidied chamber with uorescein-conjugated goat anti-mouse IgG antibody diluted 1:2000 in PBScontaining 2% BSA and 2% NGS. The sections were then washed inPBS and some sections were briey counterstained with PropidiumIodide (1 g/ml; Sigma, UK) for 5 s, collected on microscope slidesandmounted in Citiuor (AgarScientic), a uorescence-enhancingmedium. For qualitative purposes further sections were incubated for 20 min in a PBS solution containing Neuro-Trace, a uorescentNissl stain (1:100, Invitrogen), and counterstained with Hoechst33258 (1:1000, MolecularProbes, USA). Heat maps were created bypseudo-colouring photomicrographs of sections stained with the G10reelin antibody and an anti-mouse FITC-labeled secondaryantibody,areas of intense reelin immunopositive stain being represented byred.

Quantitative evaluation of reelin-positive cells. A montageof three separate images that included the entire depth of theprelimbic cortex was created using a Leica DMLB uoresc-ence microscope. A counting frame (0.898 0.349 mm 2 ), outliningthe width of each layer, was overlaid on a montage of imagesobtained from the prelimbic cortex to facilitate counting of layer-specic cell number. Seven separate montages, derived fromserial sections obtained from each animal were used to estimatereelin immunopositive cells and this number was normalised tocells/mm 2 /unit area by dividing by the area of each layer. Thelayer areas employed were Layer I: 0.052 mm 2 ; Layer II: 0.017mm 2 ; Layer III: 0.043 mm 2 ; Layer V: 0.098 mm 2 ; and Layer VI:0.101 mm 2 . Cell counts were standardised to unit area of thegranule cell layer and expressed as mean SEM of cells/mm 2 .Statistical analysis employed the Students t -test and a signi-cance level of P 0.05 was employed in all cases.

RESULTS

Developmental emergence of behavioural decits inrats reared in isolation

Analysis of open-eld behaviour in rats maintained in iso-lation from postnatal day 25 revealed signicant decits in

the ability of adult animals (postnatal day 80) to habituateto a novel environment. Animals reared in social groupsexhibited the expected decrease in locomotor activity (ses-sion 1: 171.8 7.6; session II: 91.4 13.4; P 0.05) andvertical rearing (session 1: 22.5 1.4; session II: 11.6 1.4;rears/unit time; P 0.05) upon re-exposure to the open-eldparadigm. By contrast, animals reared in isolation failed tohabituate to the open-eld environment (Locomotor activity session 1: 182.9 13.1; session II: 171.9 15.9; P 0.05) andvertical rearing (Vertical rearingsession 1: 23.4 1.1; ses-sion II: 21.43 0.9; rears/unit time; P 0.05). This failure tohabituate in the open-eld paradigm is in agreement with allprevious studies that have employed isolation rearing tomodel features of neuropsychiatric conditions (for a reviewsee Fone and Porkess, 2008 ). However, this habituationdecit did not emerge in a developmental manner as bothrearing and locomotor activity over postnatal days 3060

was found to be most erratic (data not shown). Animals reared in isolation also displayed impairments

in sensorimotor processing, as assessed using the pre-pulse inhibition paradigm. These animals tended to exhibitan increased responsiveness, as judged by their basalstartle amplitude to a single 120 dB acoustic startle stim-ulus, and this was signicantly different to that observed inthe social control group at postnatal day 30 ( P 0.0019;unpaired two-tailed Students t -test) and postnatal day 60(P 0.0193; unpaired two-tailed Students t -test) ( Fig. 1 A).During behavioural testing the calibration of the move-ment-sensitive platform was maintained at a constant leveland the observed steady increase in basal startle ampli-tude was, therefore, directly correlated with the weight gainof cohorts over development. Exposure of animals rearedin isolation or maintained in social groups to separateprepulse stimuli of 72, 76, 80 and 84 dB resulted in anincreasing inhibition of the response to a subsequent star-tle stimulus of 120 dB ( Fig. 1B). Given the small prepulseincrements employed, the response curve was shallow butrobust at all developmental timepoints examined, with theexception of that obtained at postnatal day 40. Comparisonof the cohort reared in isolation to that of the social controlgroup revealed no signicant reductions in prepulse inhi-bition when tested at postnatal day 30 ( F [1,52] 0.083;P 0.7740; two-way ANOVA) and postnatal day 40(F [1,56] 1.515; P 0.2236), however, signicant decits

became apparent at postnatal day 60 ( F [1,56] 15.38;P 0.0002) and these persisted into maturity at postnatalday 80 ( F [1,52] 17.35; P 0.0001). These effects on pre-pulse inhibition have been demonstrated to be indepen-dent of basal startle effects in all previous studies onisolation rearing ( Geyer et al., 1993; Domeney and Feldon,1998; Heidbreder et al., 2001 ).

Disruption of reelin expression in rats rearedin isolation

In order to relate the age-dependent emergence of cogni-tive decits to a cellular determinant of neural structuring,we further examined the inuence of isolation rearing on

the expression of reelin in the prefrontal cortex. Immuno-blots, developed using the G10 monoclonal antibody to

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reelin, reliably detected the 400 kDa full length protein andits expected proteolytic fragments of 300 and 180 kDa ( Fig.2 A). Strong immunoreactivity was detected in both the

400, 300 and 180 kDa reelin bands at all developmentalages examined in both the prefrontal cortex obtained from

animals reared in isolation and social groups. Semi-quan-titative analysis of these immunoblots revealed the full-length 400 kDa reelin protein in the prefrontal cortex to besignicantly modulated during the development of animalsreared in isolation, as compared to that observed in thesocial control group ( Fig. 2B). Reelin expression remainedunchanged over postnatal days 30 and 40 but exhibited a60% increase at postnatal day 60 followed by a 40%decrease at postnatal day 80 in the isolation reared ani-mals, both modulations being signicant relative to thesocial control group ( P 0.0419 and P 0.0003, respec-tively; two-tailed unpaired Students t -test).

Fig. 2. Inuence of isolation rearing on reelin expression in the pre-frontal cortex (PFC) of Wistar rats at increasing age. Immunoblotsillustrating the major protein products of the reelin protein are shown inPanel A and their semi-quantitative densitometric analysis is shown inPanel B. The values are expressed as the mean SEM and signicantdifferences (two-tailed unpaired Students t -test) between the isolationreared (IR; lled circles) and social cohorts (SC; open circles) areindicated in the gure. Signicant differences by t -test are indicatedwith an asterisk ( P 0.05).

Fig. 1. Inuence of isolation rearing on pre-pulse inhibition in Wistar rats of increasing age. Basal startle amplitude (Panel A) values areexpressed as the mean SEM and those signicantly different(P 0.05; unpaired two-tailed Students t -test) between the isolationreared group ( n 78) and the social control group ( n 78) areindicated with an asterisk. The effect of separate prepulse stimuli onstartle inhibition to a subsequent pulse of 120 dB is shown in Panel B.The values are expressed as the mean SEM and signicant differ-ences (two-way ANOVA with Bonferroni post hoc test) between theisolation reared (lled columns and circles) and social cohorts (opencolumns and circles) are indicated in the gure.



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The above isolation rearing-induced modulations inreelin expression may relate to change in the rate of pro-tein synthesis or to aberrations in the developmental ex-pression of GABA interneurons, the main source of reelin.Immunohistochemistry was, therefore, employed to exam-ine the expression of reelin-positive cells in cohorts of animals reared in isolation and in social groups. Reelin-immunopositive cells were found to be predominantly lo-cated to layers I and II of the prefrontal cortex ( Fig. 3 A)where reelin was found to be strongly expressed in thecytoplasmic compartment ( Fig. 3B). Quantitative analysisof the reelin immunopositive cells in layer I of the prefrontalcortex revealed negligible change in the density of thesecells during development of animals reared in socialgroups ( Fig. 3C). By contrast, isolation rearing induced asignicant dysregulation in the density of immunpositive

cells (Fig. 3C), in a manner that was reminiscent of thatobserved for reelin protein expression ( Fig. 2B). No differ-

ence in the density of reelin immunopositive cells wasapparent at postnatal day 40, however, their number showed a signicant increase of 40% at postnatal day 60and 50% decrease at postnatal day 80 ( P 0.0276 andP 0.0196, respectively; two-tailed unpaired Students t -test). This effect was restricted to layer I of the prefrontalcortex as no signicant change in reelin immunopositivecell frequency in layers II-VI was induced by isolationrearing ( Table 1 ).

Closer inspection of the reelin immunopositive cells inlayer I of the prefrontal cortex allowed the identication of two sub-populations of cells based on the alignment of their immunoreactivity pattern and their position withinlayer I. Cells expressing reelin immunoreactivity in a hori-zontal orientation were more numerous in the upper re-gions of layer I, as evidenced by the few counterstained

propidium iodide nuclei, suggesting these to be the bipolar cells (Fig. 4 A, B) (Bacon et al., 1996; Gabbott et al., 1997 ).

I

II

III

V

VI

Deeplayers

Upper layers

piaA

20 m

B

*

*

R e e

l i n p o s

i t i v e c e

l l s

/ m m

2

60

80

100

120

140

160

180

200

Postnatal day40 60 80

C

Layer I

Fig. 3. Inuence of isolation rearing on the density of reelin-expressing cells in the prefrontal cortex of Wistar rats at increasing age. The distributionof immunopositive cells in the layers of the prefrontal cortex is illustrated in Panel A and the extra-nuclear location of the immunoreactivity is shownin Panel B (red demonstrating highest expression). Quantitation of cell density in layer I of the prefrontal cortex is shown in Panel C and values areexpressed as the mean SEM and signicant differences between the isolation reared (lled circles) and social cohorts (open circles) are shown withan asterisk ( P 0.05, two-tailed unpaired Students t -test). For interpretation of the references to color in this gure legend, the reader is referred tothe Web version of this article.



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Those with reelin immunoreactivity exhibiting a verticalorientation were more numerous in the lower regions of layer I, where a greater number of counterstained nucleiwere evident ( Fig. 4C, D), suggesting these to be thevertical cells whose arbors extend into cortical layers II andIII (Fig. 4E, F) (Gabbott et al., 1997 ). Following isolationrearing, both horizontal and vertical cell populations weresignicantly decreased at postnatal day 80 ( P 0.0100 andP 0.0415, respectively, two-tailed unpaired Students t -test), however, the signicant increase in overall reelin-positive cell number at postnatal day 60 ( Fig. 3) was ob-served only in the vertical cell population ( P 0.0348, two-tailed unpaired Students t -test) ( Fig. 4G, H).

DISCUSSIONDevelopmental emergence of isolationrearing-induced decits in prepulse inhibition

Rearing Wistar rats in isolation from time of weaning re-sulted in hyperlocomotion and signicant decits in pre-pulse inhibition, as has been previously reported in similar studies ( Geyer et al., 1993; Fone and Porkess, 2008 ) andargued to be hallmarks of the behavioural decits associ-ated with neuropsychiatric conditions ( Swerdlow et al.,1994 ). The inability of rats reared in isolation to habituateto the open-eld environment demonstrates disrupted in-tegration of past and current experience, which is believed

to be a core decit contributing to acute psychosis ( Gray,1998; Gray et al., 1999 ). Moreover, hyperlocomotion anddecits in sensorimotor processing observed in animalsreared in isolation has been related to increased subcorti-cal dopamine transmission ( Swerdlow et al., 2001 ). Im-paired prepulse inhibition is thought to reect the cognitivefragmentation associated with such conditions ( Braff andGeyer, 1990; Geyer et al., 1993 ), and, in close alignmentwith the human conditions, these decits emerge duringadolescence in isolation reared rats ( Lipska et al., 1995;Bakshi and Geyer, 1999 ). In conclusion, the spectrum of behavioural abnormalities observed in rats reared in iso-lation further validate this model as one that reasonably

recapitulates some of the major correlates of neuropsychi-atric disorders.

Correlation of reelin expression with prepulseinhibition decits

The possible association of impaired neuroplastic mecha-nisms with the developmental emergence of cognitive def-icits in animals reared in isolation was provided by ananalysis of reelin expression. In the prefrontal cortex, thematching increase in reelin protein expression and fre-quency of reelin immunopositive cells in layer I suggestedthe emergence of prepulse inhibition decits at postnatalday 60 to be accompanied by a signicant increase in theproduction of this extracellular matrix protein. Within layer I,which consists almost entirely of GABAergic interneurons,the classic horizontal interneurons extend their wide dendriticarbour throughout this layer ( Hestrin and Armstrong, 1996;

Gabbott et al., 1997 ). By contrast, the vertical GABAergicinterneurons within layer I project descending axons intodeeper cortical lamina of layers II and III ( Gabbott et al., 1997; Aguil et al., 1999 ). Interestingly, the timing of the increasein reelin expression coincides with the proliferation of amygdalar and ventral hippocampal afferents on tothese GABAergic interneurons and pyramidal neuronsof layers II and III during late adolescence in rodents,primates and humans ( Huttenlocher and Dabholkar, 1997;Gogtay et al., 2004; Cunningham et al., 2002, 2008 ). Asthe role of this secreted matrix protein in later postnatalperiods relates mainly to dendritic remodelling and syn-aptogenesis, the marked change in reelin expression ob-served at postnatal day 60 in isolation reared animals sug-gests the emergence of prepulse inhibition decits maybeassociated with enhanced, possibly excessive synapse re-modelling (Jay and Witter, 1991 ; Bacon et al., 1996 ; Borrel etal., 1999; Costa et al., 2001; Dong et al., 2003; Cunninghamet al., 2002, 2008; Boqoch and Linial, 2008 ).

The persistence of decits in prepulse inhibition andthe decrease in reelin expression at postnatal day 80further suggests that the reelin-associated changes maybe initially compensatory in nature but fail to correct theisolation-induced dysregulation of the developing neuralcircuits, the low reelin expression ultimately resulting insynapse loss. This concept is consistent with post-mor-tem studies on tissue derived from schizophrenic pa-

tients that have demonstrated reduced neuropil and syn-apse number in layer III ( Selemon and Goldman-Rakic,

Table 1. Expression of reelin immunopositive cells in individual layers of the prelimbic cortex in isolation-reared and social control groups

Layer I Layer II Layer III Layer V Layer VI

Postnatal day 40Social control 148.4 12.18 94.54 33.15 84.72 15.49 94.02 19.46 65.77 13.65Isolation reared 151.8 14.85 92.44 22.76 90.53 7.58 95.12 14.29 64.36 4.64

Postnatal day 60Social control 138.7 7.31 119.7 5.29 69.77 9.00 70.34 6.85 63.65 8.65Isolation reared 179.4 12.00* 102.2 2.43 82.78 6.17 70.70 4.93 61.23 4.85

Postnatal day 80Social control 136.7 8.11 98.74 16.94 68.94 10.45 72.89 6.35 55.16 9.83Isolation reared 87.91 13.13* 60.92 12.55 47.34 8.72 56.85 12.89 48.80 15.00

Data represents the mean SEM (n 34) of reelin immunopositive cells/mm 2 in each layer of the prelimbic cortex. Values signicantly different fromthe social control group are indicated with an asterisk (*)( P 0.05; two-tailed unpaired Students t -test).



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1999 ) and the substantial losses of GABAergic interneu-ron markers in layers IIII of the medial prefrontal cortex(Glantz and Lewis, 1997; Day-Wilson et al., 2006;

Bloomeld et al., 2008; Woo et al., 1998; Pierri et al.,1999; Volk et al., 2002 ).

CONCLUSION

In conclusion, our observations on the expression of reelin-

positive cells accompanying the developmental emer-gence of behavioural decits during isolation rearing may

Merged Reelin 20 m

Merged Reelin 20 m

Reelin-expressinghorizontal cells

Reelin-expressingvertical cells

A B

C D

Layer I

Layer II

Reelin-expressing

vertical cell

F


*20

40

60

80

100

R e e

l i n p o s

i t i v e c e

l l s

/ m m

2

120

GReelin-expressing

horizontal cells

*

*

20

40

60

80

100

R e e

l i n p o s

i t i v e c e

l l s / m

m 2

120


HReelin-expressing

vertical cells

Fig. 4. Inuence of isolation rearing on the density of two separate reelin-expressing cell populations in layer I of the prefrontal cortex of Wistar ratsat increasing age. Cells expressing reelin immunoreactivity in a horizontal and vertical manner are shown in Panels AD and the position of the verticalcells in relation to layer II is shown in Panels E and F. The reelin-expressing cells in Panels A, C and F are counter-stained with NeuroTraceuorescent Nissl stain and Hoechst 33258. Panel E is a phase contrast image. Quantitation of two separate reelin-expressing cell populations in layer I of the prefrontal cortex is shown in Panels G and H and values are expressed as the mean SEM and signicant differences between the isolationreared (lled circles) and social cohorts (open circles) are shown with an asterisk ( P 0.05, two-tailed unpaired Students t -test). For interpretation of the references to color in this gure legend, the reader is referred to the Web version of this article.



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provide a basis for the structural decits observed in neu-ropsychiatric conditions, however, as yet, the physiologicaltrigger that leads to reelin over-expression at postnatal day60 remains to be established in this animal model.

AcknowledgmentsThe Applied Neurotherapeutics ResearchGroup is a Strategic Research Cluster funded jointly by ScienceFoundation Ireland (07/IN.1/B1322) and Wyeth Discovery.

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(Accepted 16 December 2009)(Available online 24 December 2009)


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