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Neuroscience 166 (2010) 377385

DEVELOPMENTAL EMERGENCE OF REELIN DEFICITS IN THE PREFRONTAL CORTEX OF WISTAR RATS REARED IN SOCIAL ISOLATION
A. W. CASSIDY,a S. K. MULVANY,a M. N. PANGALOS,b K. J. MURPHYa AND C. M. REGANa*
a The Applied Neurotherapeutics Research Group, School of Biomolecular 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 many neuropsychiatric disorders relate to GABAergic interneuron structure and function, we employed isolation rearing of Wistar rats as a model to correlate developmental emergence of cognitive decits with the expression of reelin-producing interneurons in the medial prefrontal cortex (PFC). Prepulse inhibition decits emerged at postnatal day 60 and persisted into adulthood. Paralleling the emergence of these neurobehavioural decits was an increase in reelin production and reelin-immunopositive cells in layer I of the PFC and this later became signicantly reduced at postnatal day 80. Cells expressing reelin immunoreactivity in a horizontal orientation were mainly located to the upper regions of layer I whereas those with a vertical orientation, whose arbors extend into cortical layers II and III, were more numerous in the lower regions of layer I and became signicantly dysregulated during postnatal development. No behavioural decits or altered reelin expression was observed at postnatal days 30 or 40. Developmental emergence of neurobehavioural and reelin decits in isolation reared animals is proposed to reect maladaptive wiring within the medial prefrontal cortex during a critical maturation period of this circuitry. 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: prepulse inhibition, GABAergic interneurons, isolation rearing, layer I, immunouorescence, prelimbic cortex.

Studies of postmortem brain tissue have provided signicant evidence that the GABAergic system is signicantly disrupted in a number of neuropsychiatric disorders (Lewis et al., 2005; Lisman et al., 2008). Such studies have shown a reduction in protein markers of interneuron function in post-mortem tissue obtained from the prefrontal cortex (Akbarian et al., 1995; Beasley and Reynolds, 1997; Sakai et al., 2008), cingulate cortex (Benes et al., 1991; Woo et al., 2004; Oblak et al., 2009), and temporal lobe (Chance et al., 2005). The reproducibility of these ndings supports
*Corresponding author. Tel: 353-1-716-6775; fax: 353-1-716-6920. E-mail address: ciaran.regan@ucd.ie (C. M. Regan). Abbreviations: ANOVA, analysis of variance; BSA, bovine serum albumin; EDTA, ethylenediamine tetra acetic acid; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; NGS, normal goat serum; PBS, phosphate-buffered saline; PFC, prelimbic region of prefrontal cortex; PPI, prepulse inhibition; TBS-T, tris-buffered saline.

the possibility that interneuron decits are a central feature in 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 and secrete reelin (Pesold et al., 1998, 1999), a large extracellular matrix glycoprotein (400 kDa) that has a wide array of functions in both the developing and adult cortex. During early development reelin is synthesised by CajalRetzius interneurons and plays a vital role in the lamination of cortical cell layers (Tissir and Gofnet, 2003). In adulthood reelin becomes more widely expressed by GABA interneurons, 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 300 350 amino acids and, upon secretion into the extracellular space, is cleaved by metalloproteinases between repeats 23 and 6 7, the central 3 6 repeats being required for activation of 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 space surrounding dendrites, dendritic spines and axon boutons (DArcangelo et al., 1995; Alcntara et al., 1998; Pappas et al., 2002; Tissir and Gofnet, 2003) is known to regulate synapse structure and stability (Weeber et al., 2002; Dong et al., 2003). Evidence exists to implicate reelin decits in the developmental emergence of a number of neuropsychiatric conditions including schizophrenia, bipolar disorder (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 compromised as both reelin mRNA and GAD67 mRNA expression have been found to be signicantly depressed in GABAergic interneurons in the supercial layers of the prefrontal cortex (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 activity may, in part, be responsible for the emergence of this and other psychotic states, however, postmortem tissue derived from patients at the end-stage of schizophrenia cannot provide an insight into the preceding developmental mechanisms. As many psychiatric disorders emerge during late adolescence (Paus et al., 2008), animal models that recapitulate their cardinal features in adulthood become a priority in any attempt to understand the developmental mecha-

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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A. W. Cassidy et al. / Neuroscience 166 (2010) 377385 periods in which there was no prepulse or startle stimulus. The session terminated with an additional ve startle trials. Signals were 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 determined in separate cohorts of animals (n7 8) on postnatal days 30, 40, 60 and 80. The effects of isolation rearing on pre-pulse inhibition, as compared to that of the aged-matched cohorts (n7 8) of social animals, were assessed by two-way analysis of variance (ANOVA) with post hoc analysis using a Bonferroni post t-test. In all cases, P-values less than 0.05 were considered to be signicant.

nisms that lead to the onset of psychosis. Several models have been extensively studied and include repetitive administrations 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). In each model, the behavioural phenotype includes hyperlocomotion and prepulse inhibition decits. Most of these abnormalities emerge during adolescence and in many cases there is also evidence of a reduction in markers of GABAergic interneurons (Lipska et al., 2003; Pillai-Nair et al., 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 behavioural responses, we have correlated alteration of reelin protein expression and reelin-secreting cells with emergence of behavioural decits in isolation reared animals.

Immunoblot analysis of reelin expression


Tissue preparation. Separate cohorts (n4) of naive animals reared in isolation or social groups were killed on postnatal days 30, 40, 60 and 80, the brain removed and the medial prefrontal cortex dissected and excised. Samples were immediately placed in cryotubes, snap frozen in liquid nitrogen and stored in a 80 C freezer until required. Immediately prior to use, the samples were homogenised at 4 C in 300 l of 10 mM HEPES, pH 7.4, containing 0.32 M sucrose, 2 mM EDTA, and 0.01% of a protease and phosphatase inhibitor cocktail (Sigma, UK). The homogenates were subsequently centrifuged (1000 rpm, 15 min) and the supernatant was removed and stored at 20 C until further use. SDS polyacrylamide gel electrophoresis and Western-blotting procedure. Protein concentrations were determined by the BCA 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 33 mM NaCl, 1 mM EDTA, 2% (w/v) SDS, 0.01% (w/v) Bromophenol Blue, 10% glycerol and 3% v/v dithiothreitol reducing agent. The reduced and solubilised proteins were separated using pre-prepared 5% polyacrylamide gels and, subsequently, transferred to nitrocellulose membranes, according to manufacturers instructions (Biorad, UK). Pre-stained molecular weight markers were co-electrophoresed with the protein samples (Sigma, UK). Successful protein transfer was conrmed by staining the nitrocellulose sheet with Ponceau Red solution (Sigma, UK) prior to immunoblotting and by Napthol Blue (Sigma, UK) following completion of immunoblotting. Reactive groups on the nitrocellulose sheet were then inactivated using Tris buffered saline solution (TBS-T) blocking buffer (10 mM TrisHCl, pH 7.4, containing 150 mM NaCl, 0.05% (v/v) Tween-20, and with 5% (w/v) non-fat milk powder) for 1 h at room temperature. The membrane was subsequently 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, the nitrocellulose membrane was washed three times in TBS-T pH 7.4 before being incubated for 1 h in blocking buffer containing a horse radish peroxidase-conjugated anti-mouse IgG monoclonal antibody (Novagen, UK; 1:20,000 dilution). After incubation, the nitrocellulose 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 optimal resolution of the protein bands was achieved. Immunoblot analysis of Reelin with the G10 antibody revealed three specic protein bands, the full length 400 kDa protein and two bands at 300 and 180 kDa, the latter representing the proteolytic fragments generated by metalloproteinase cleavage at repeats 23 and 6 7, respectively. The X-ray lms were scanned, converted into a digital format and the immunostained band density analysed using ImageJ software (http://rsb.info.nih.gov/ij/docs/index.html). The Naphthol Black-stained cellulose sheet was also scanned and digitised and used to correct the immunostained bands for unequal protein loading.

EXPERIMENTAL PROCEDURES
Animal maintenance
Experimentally naive male Wistar rats were employed in all studies. The animals were purpose bred at the Biomedical Facility, University College Dublin, and maintained in standard laboratory conditions until the time of experimental use. Animals were introduced to the experimental holding rooms 5 days prior to the commencement of the study, housed in groups of 3 4 during this period, and maintained at 2224 C on a standard 12 h light/dark cycle, with food and water available ad libitum. On each of the 2 days preceding commencement of behavioural studies, the animals were handled and weighed and assessed in an open-eld arena for locomotor activity, rearing and general behaviour over a 5 min period. All observations were carried out in the quiet room under low-level, red light illumination between 8:00 and 12:00 h to minimise the inuence of circadian rhythms. Isolation-reared animals (isolated animals) were housed individually in non-soft bottom cages (225345170 mm), from time of weaning (postnatal day 25) until completion of behavioural testing. The standard 12 h light/dark cycle was maintained and food and water was provided ad libitum. Noise and visual stimuli were kept to an absolute minimum as previously described (Geyer et al., 1993). All experimental procedures were approved by the Animal Research Ethics Committee of University College Dublin, conformed to EU Council Directive 86 609-EEC, and were carried out by individuals retaining the appropriate licence issued by the Irish Department of Health. Throughout the course of these studies, every attempt was 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 sensorimotor gating decits (Swerdlow et al., 1994) and the protocol employed was based on a procedure previously described by Geyer and colleagues (1993). Each rat was restrained in an appropriately sized cylindrical holder, placed on a movementsensitive platform and maintained in a soundproof chamber. The rat was allowed to habituate to a white noise background of 70 dB for 5 min before receiving ve 20 ms startle trials of 120 dB, separated by randomised intervals of 10 20 s. Immediately thereafter, each rat received ve separate presentations with one of the prepulse stimuli of 72, 76, 80, or 84 dB and these were followed, 100 ms later, by the 120 dB acoustic startle stimulus. Each trial was separated by a time interval of 10 20 s. The four prepulse stimuli were delivered in a randomised manner and included

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Quantitative immunohistochemical analysis of reelin expression in vivo


Tissue preparation. Animals were terminally anaesthetised using 1 ml/kg sodium pentobarbital (Euthatal, Pzer Animal Health, UK) and their tissue was xed by transcardial perfusion with a saline solution (0.9% w/v) for 2 min, followed by a 20 min perfusion with 0.12 M Srenson phosphate buffer (pH 7.2) containing 4% paraformaldehyde. The animals were then killed and their brains were removed and stored in 4% paraformaldehyde in Srensons buffer for a 24 h period. Following xation, the brains were placed in a cryoprotective solution (40% sucrose [w/v] and 10% glycerol [v/v] in dH20) at 4 C for approximately 48 h. The brains were then coated in optimum cutting temperature compound (O.C.T.; Tissue-Tek, UK), to provide an even freezing rate, and lowered into a Cryoprep freezing apparatus (Algen Inc, UK) containing liquid CO2 cooled n-hexane. The tissue was stored at 80 C until required. Cryosectioning and immunohistochemical protocols. Sections (16 m thick) of the prefrontal cortex were taken at level 3.2 mm rostral to bregma (Paxinos and Watson, 1986) and placed in 0.32 M sucrose solution for 5 min. Free-oating sections were then washed twice in 0.1 M (pH 7.4) PBS solution (phosphate buffered saline; Sigma, UK) solution for 10 min. Subsequently, the sections were incubated 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 PBS containing 2% NGS and 2% bovine serum albumen (BSA; Sigma, UK). The sections were then washed twice in PBS for 10 min and further incubated for 3 h in the humidied chamber with uoresceinconjugated goat anti-mouse IgG antibody diluted 1:2000 in PBS containing 2% BSA and 2% NGS. The sections were then washed in PBS and some sections were briey counterstained with Propidium Iodide (1 g/ml; Sigma, UK) for 5 s, collected on microscope slides and mounted in Citiuor (Agar Scientic), a uorescence-enhancing medium. For qualitative purposes further sections were incubated for 20 min in a PBS solution containing Neuro-Trace, a uorescent Nissl stain (1:100, Invitrogen), and counterstained with Hoechst 33258 (1:1000, Molecular Probes, USA). Heat maps were created by pseudo-colouring photomicrographs of sections stained with the G10 reelin antibody and an anti-mouse FITC-labeled secondary antibody, areas of intense reelin immunopositive stain being represented by red. Quantitative evaluation of reelin-positive cells. A montage of three separate images that included the entire depth of the prelimbic cortex was created using a Leica DMLB uorescence microscope. A counting frame (0.8980.349 mm2), outlining the width of each layer, was overlaid on a montage of images obtained from the prelimbic cortex to facilitate counting of layerspecic cell number. Seven separate montages, derived from serial sections obtained from each animal were used to estimate reelin immunopositive cells and this number was normalised to cells/mm2/unit area by dividing by the area of each layer. The layer areas employed were Layer I: 0.052 mm2; Layer II: 0.017 mm2; Layer III: 0.043 mm2; Layer V: 0.098 mm2; and Layer VI: 0.101 mm2. Cell counts were standardised to unit area of the granule cell layer and expressed as meanSEM of cells/mm2. Statistical analysis employed the Students t-test and a signicance level of P0.05 was employed in all cases.

the ability of adult animals (postnatal day 80) to habituate to a novel environment. Animals reared in social groups exhibited the expected decrease in locomotor activity (session 1: 171.87.6; session II: 91.413.4; P0.05) and vertical rearing (session 1: 22.51.4; session II: 11.61.4; rears/unit time; P0.05) upon re-exposure to the open-eld paradigm. By contrast, animals reared in isolation failed to habituate to the open-eld environment (Locomotor activity session 1: 182.913.1; session II: 171.915.9; P0.05) and vertical rearing (Vertical rearingsession 1: 23.41.1; session II: 21.430.9; rears/unit time; P0.05). This failure to habituate in the open-eld paradigm is in agreement with all previous studies that have employed isolation rearing to model features of neuropsychiatric conditions (for a review see Fone and Porkess, 2008). However, this habituation decit did not emerge in a developmental manner as both rearing and locomotor activity over postnatal days 30 60 was found to be most erratic (data not shown). Animals reared in isolation also displayed impairments in sensorimotor processing, as assessed using the prepulse inhibition paradigm. These animals tended to exhibit an increased responsiveness, as judged by their basal startle amplitude to a single 120 dB acoustic startle stimulus, and this was signicantly different to that observed in the social control group at postnatal day 30 (P0.0019; unpaired two-tailed Students t-test) and postnatal day 60 (P0.0193; unpaired two-tailed Students t-test) (Fig. 1A). During behavioural testing the calibration of the movement-sensitive platform was maintained at a constant level and the observed steady increase in basal startle amplitude was, therefore, directly correlated with the weight gain of cohorts over development. Exposure of animals reared in isolation or maintained in social groups to separate prepulse stimuli of 72, 76, 80 and 84 dB resulted in an increasing inhibition of the response to a subsequent startle stimulus of 120 dB (Fig. 1B). Given the small prepulse increments employed, the response curve was shallow but robust at all developmental timepoints examined, with the exception of that obtained at postnatal day 40. Comparison of the cohort reared in isolation to that of the social control group revealed no signicant reductions in prepulse inhibition when tested at postnatal day 30 (F[1,52]0.083; P0.7740; two-way ANOVA) and postnatal day 40 (F[1,56]1.515; P0.2236), however, signicant decits became apparent at postnatal day 60 (F[1,56]15.38; P0.0002) and these persisted into maturity at postnatal day 80 (F[1,52]17.35; P0.0001). These effects on prepulse inhibition have been demonstrated to be independent of basal startle effects in all previous studies on isolation rearing (Geyer et al., 1993; Domeney and Feldon, 1998; Heidbreder et al., 2001). Disruption of reelin expression in rats reared in isolation In order to relate the age-dependent emergence of cognitive 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. Immunoblots, developed using the G10 monoclonal antibody to

RESULTS
Developmental emergence of behavioural decits in rats reared in isolation Analysis of open-eld behaviour in rats maintained in isolation from postnatal day 25 revealed signicant decits in

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animals reared in isolation and social groups. Semi-quantitative analysis of these immunoblots revealed the fulllength 400 kDa reelin protein in the prefrontal cortex to be signicantly modulated during the development of animals reared in isolation, as compared to that observed in the social control group (Fig. 2B). Reelin expression remained unchanged over postnatal days 30 and 40 but exhibited a 60% increase at postnatal day 60 followed by a 40% decrease at postnatal day 80 in the isolation reared animals, both modulations being signicant relative to the social control group (P0.0419 and P0.0003, respectively; two-tailed unpaired Students t-test).

Fig. 1. Inuence of isolation rearing on pre-pulse inhibition in Wistar rats of increasing age. Basal startle amplitude (Panel A) values are expressed as the meanSEM and those signicantly different (P0.05; unpaired two-tailed Students t-test) between the isolation reared group (n7 8) and the social control group (n7 8) are indicated with an asterisk. The effect of separate prepulse stimuli on startle inhibition to a subsequent pulse of 120 dB is shown in Panel B. The values are expressed as the meanSEM and signicant differences (two-way ANOVA with Bonferroni post hoc test) between the isolation reared (lled columns and circles) and social cohorts (open columns and circles) are indicated in the gure.

reelin, reliably detected the 400 kDa full length protein and its expected proteolytic fragments of 300 and 180 kDa (Fig. 2A). Strong immunoreactivity was detected in both the 400, 300 and 180 kDa reelin bands at all developmental ages examined in both the prefrontal cortex obtained from

Fig. 2. Inuence of isolation rearing on reelin expression in the prefrontal cortex (PFC) of Wistar rats at increasing age. Immunoblots illustrating the major protein products of the reelin protein are shown in Panel A and their semi-quantitative densitometric analysis is shown in Panel B. The values are expressed as the meanSEM and signicant differences (two-tailed unpaired Students t-test) between the isolation reared (IR; lled circles) and social cohorts (SC; open circles) are indicated in the gure. Signicant differences by t-test are indicated with an asterisk (P0.05).

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pia

Upper layers

I II
20 m

III
Reelin positive cells/mm2

200

180 160

140

Deep layers

120

100

Layer I

80

VI

60

40

60

80

Postnatal day
Fig. 3. Inuence of isolation rearing on the density of reelin-expressing cells in the prefrontal cortex of Wistar rats at increasing age. The distribution of immunopositive cells in the layers of the prefrontal cortex is illustrated in Panel A and the extra-nuclear location of the immunoreactivity is shown in Panel B (red demonstrating highest expression). Quantitation of cell density in layer I of the prefrontal cortex is shown in Panel C and values are expressed as the meanSEM and signicant differences between the isolation reared (lled circles) and social cohorts (open circles) are shown with an asterisk (P0.05, 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.

The above isolation rearing-induced modulations in reelin expression may relate to change in the rate of protein synthesis or to aberrations in the developmental expression of GABA interneurons, the main source of reelin. Immunohistochemistry was, therefore, employed to examine the expression of reelin-positive cells in cohorts of animals reared in isolation and in social groups. Reelinimmunopositive cells were found to be predominantly located to layers I and II of the prefrontal cortex (Fig. 3A) where reelin was found to be strongly expressed in the cytoplasmic compartment (Fig. 3B). Quantitative analysis of the reelin immunopositive cells in layer I of the prefrontal cortex revealed negligible change in the density of these cells during development of animals reared in social groups (Fig. 3C). By contrast, isolation rearing induced a signicant dysregulation in the density of immunpositive cells (Fig. 3C), in a manner that was reminiscent of that observed for reelin protein expression (Fig. 2B). No differ-

ence in the density of reelin immunopositive cells was apparent at postnatal day 40, however, their number showed a signicant increase of 40% at postnatal day 60 and 50% decrease at postnatal day 80 (P0.0276 and P0.0196, respectively; two-tailed unpaired Students ttest). This effect was restricted to layer I of the prefrontal cortex as no signicant change in reelin immunopositive cell frequency in layers II-VI was induced by isolation rearing (Table 1). Closer inspection of the reelin immunopositive cells in layer 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 within layer I. Cells expressing reelin immunoreactivity in a horizontal orientation were more numerous in the upper regions of layer I, as evidenced by the few counterstained propidium iodide nuclei, suggesting these to be the bipolar cells (Fig. 4A, B) (Bacon et al., 1996; Gabbott et al., 1997).

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Table 1. Expression of reelin immunopositive cells in individual layers of the prelimbic cortex in isolation-reared and social control groups Layer I Postnatal day 40 Social control Isolation reared Postnatal day 60 Social control Isolation reared Postnatal day 80 Social control Isolation reared Layer II Layer III Layer V Layer VI

148.412.18 151.814.85 138.77.31 179.412.00* 136.78.11 87.9113.13*

94.5433.15 92.4422.76 119.75.29 102.22.43 98.7416.94 60.9212.55

84.7215.49 90.537.58 69.779.00 82.786.17 68.9410.45 47.348.72

94.0219.46 95.1214.29 70.346.85 70.704.93 72.896.35 56.8512.89

65.7713.65 64.364.64 63.658.65 61.234.85 55.169.83 48.8015.00

Data represents the meanSEM (n3 4) of reelin immunopositive cells/mm2 in each layer of the prelimbic cortex. Values signicantly different from the social control group are indicated with an asterisk (*)(P0.05; two-tailed unpaired Students t-test).

Those with reelin immunoreactivity exhibiting a vertical orientation were more numerous in the lower regions of layer I, where a greater number of counterstained nuclei were evident (Fig. 4C, D), suggesting these to be the vertical cells whose arbors extend into cortical layers II and III (Fig. 4E, F) (Gabbott et al., 1997). Following isolation rearing, both horizontal and vertical cell populations were signicantly decreased at postnatal day 80 (P0.0100 and P0.0415, respectively, two-tailed unpaired Students ttest), however, the signicant increase in overall reelinpositive cell number at postnatal day 60 (Fig. 3) was observed only in the vertical cell population (P0.0348, twotailed unpaired Students t-test) (Fig. 4G, H).

Correlation of reelin expression with prepulse inhibition decits The possible association of impaired neuroplastic mechanisms with the developmental emergence of cognitive deficits in animals reared in isolation was provided by an analysis of reelin expression. In the prefrontal cortex, the matching increase in reelin protein expression and frequency of reelin immunopositive cells in layer I suggested the emergence of prepulse inhibition decits at postnatal day 60 to be accompanied by a signicant increase in the production of this extracellular matrix protein. Within layer I, which consists almost entirely of GABAergic interneurons, the classic horizontal interneurons extend their wide dendritic arbour throughout this layer (Hestrin and Armstrong, 1996; Gabbott et al., 1997). By contrast, the vertical GABAergic interneurons within layer I project descending axons into deeper cortical lamina of layers II and III (Gabbott et al., 1997; Aguil et al., 1999). Interestingly, the timing of the increase in reelin expression coincides with the proliferation of amygdalar and ventral hippocampal afferents on to these GABAergic interneurons and pyramidal neurons of 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). As the role of this secreted matrix protein in later postnatal periods relates mainly to dendritic remodelling and synaptogenesis, the marked change in reelin expression observed at postnatal day 60 in isolation reared animals suggests the emergence of prepulse inhibition decits maybe associated with enhanced, possibly excessive synapse remodelling (Jay and Witter, 1991; Bacon et al., 1996; Borrel et al., 1999; Costa et al., 2001; Dong et al., 2003; Cunningham et al., 2002, 2008; Boqoch and Linial, 2008). The persistence of decits in prepulse inhibition and the decrease in reelin expression at postnatal day 80 further suggests that the reelin-associated changes may be initially compensatory in nature but fail to correct the isolation-induced dysregulation of the developing neural circuits, the low reelin expression ultimately resulting in synapse loss. This concept is consistent with post-mortem studies on tissue derived from schizophrenic patients that have demonstrated reduced neuropil and synapse number in layer III (Selemon and Goldman-Rakic,

DISCUSSION
Developmental emergence of isolation rearing-induced decits in prepulse inhibition Rearing Wistar rats in isolation from time of weaning resulted in hyperlocomotion and signicant decits in prepulse inhibition, as has been previously reported in similar studies (Geyer et al., 1993; Fone and Porkess, 2008) and argued to be hallmarks of the behavioural decits associated with neuropsychiatric conditions (Swerdlow et al., 1994). The inability of rats reared in isolation to habituate to the open-eld environment demonstrates disrupted integration 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 and decits in sensorimotor processing observed in animals reared in isolation has been related to increased subcortical dopamine transmission (Swerdlow et al., 2001). Impaired prepulse inhibition is thought to reect the cognitive fragmentation associated with such conditions (Braff and Geyer, 1990; Geyer et al., 1993), and, in close alignment with the human conditions, these decits emerge during adolescence in isolation reared rats (Lipska et al., 1995; Bakshi and Geyer, 1999). In conclusion, the spectrum of behavioural abnormalities observed in rats reared in isolation further validate this model as one that reasonably recapitulates some of the major correlates of neuropsychiatric disorders.

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Reelin-expressing horizontal cells

G
120

Reelin-expressing horizontal cells

Reelin positive cells/mm2

100

80

Merged Reelin-expressing vertical cells

Reelin

20 m

60

40

*
20 40 60 80

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H
Merged Reelin 20 m
120

Reelin-expressing vertical cells

Reelin positive cells/mm2

Reelin-expressing vertical cell Layer I

100

80

*
60

40

Layer II
20 40 60 80

Postnatal day
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 rats at increasing age. Cells expressing reelin immunoreactivity in a horizontal and vertical manner are shown in Panels AD and the position of the vertical cells 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 NeuroTrace uorescent 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 meanSEM and signicant differences between the isolation reared (lled circles) and social cohorts (open circles) are shown with an asterisk (P0.05, 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.

1999) and the substantial losses of GABAergic interneuron 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 reelinpositive cells accompanying the developmental emergence of behavioural decits during isolation rearing may

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

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