Acta Neuropsychiatrica 2012: 24: 286–295All rights reservedDOI: 10.1111/j.1601-5215.2011.00627.x

© 2011 John Wiley & Sons A/S

ACTA NEUROPSYCHIATRICA

Neurochemical differences in two rat strainsexposed to social isolation rearing

Trabace L, Zotti M, Colaianna M, Morgese MG, Schiavone S, Tucci P,Harvey BH, Wegener G, Cuomo V. Neurochemical differences in two ratstrains exposed to social isolation rearing.

Objective: Isolation rearing of rats provides a non-pharmacologicalmethod of inducing behavioural changes in rodents that resembleschizophrenia or depression. Nevertheless, results are variable withindifferent strains. We focused on neurochemical changes in several in vivoand post-mortem brain regions of Wistar (W) and Lister Hooded (LH) ratsfollowing post-weaning social separation.Methods: Experiments were conducted after 6–8 weeks of isolation. Forpost-mortem studies, prefrontal cortex (PFC), nucleus accumbens (NAC),hippocampus (Hipp) and striatum (St) were collected by tissue dissection.In vivo experiments were conducted by microdialysis in the PFC. Analysesof dopamine (DA), serotonin (5-HT) levels and relative turnover wereperformed by using high-performance liquid chromatography.Results: We found significant strain-related differences in biogenic aminecontent. LH rats were characterised by markedly raised DA, along with itsturnover reduction, in all the post-mortem brain regions examined as wellas in microdialysis samples, while in W rats 5-HT tissue concentrationwas lower in PFC and St and higher in NAC and Hipp. Corticalextracellular 5-HT concentrations were increased in group housed anddecreased in isolated W animals. Moreover, isolation increased DAconcentrations in the PFC of LH rats, and decreased 5-HT in W rats inNAC and Hipp. Lately, 5-HT turnover was also affected by both strainand isolation conditions.Conclusions: This study suggests that W and LH rats have markedlydifferent neurochemical profiles in response to isolation, resulting inaltered monoamine levels that vary according to brain area and rat strain.These findings highlight the importance of selecting an appropriate ratstrain when considering isolation rearing to model symptoms ofschizophrenia and/or depression.

Luigia Trabace1, MargheritaZotti1, Marilena Colaianna1,Maria G. Morgese1, StefaniaSchiavone1,2,3, Paolo Tucci1,Brian H. Harvey4,∗, GregersWegener5,∗, Vincenzo Cuomo6

1Department of Biomedical Sciences, University ofFoggia, Foggia, Italy; 2Department of Pathology andImmunology, University of Geneva, Geneva,Switzerland; 3Department of Genetic and LaboratoryMedicine, Geneva University Hospitals, Geneva,Switzerland; 4Unit for Drug Research andDevelopment, Division of Pharmacology, School ofPharmacy, North-West University, Potchefstroom,South Africa; 5Centre for Psychiatric Research,University of Aarhus, Aarhus, Denmark; and6Department of Human Physiology andPharmacology, Vittorio Erspamer, University of Rome‘‘La Sapienza’’, Rome, Italy

∗These authors have contributed equally to thestudies presented in this article.

Keywords: dopamine and serotonin; isolationrearing; Lister Hooded; Wistar

Luigia Trabace, PhD, Department of BiomedicalSciences, Faculty of Medicine c/o OO.RR.,University of Foggia, Viale L. Pinto, 71100Foggia, Italy.Tel: +39 0881 588056;Fax: +39 0881 712366;E-mail: trabace@unifg.it

Accepted for publication September 29, 2011

Significant outcomes

• Different postnatal experiences have profound effects on adult neurochemistry, although the effectsvaried among strain, neurotransmitters and brain area examined.

• The neurochemical phenotype of Lister Hooded (LH) rats suggests that this strain may be morevulnerable to develop schizophrenic-like manifestations following isolation.

• The Wistar (W) rat strain could provide better information on several parameters resembling depressive-like behaviours in schizophrenia.

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Limitations

• Animal models of mental disorders are difficult to assess, especially for schizophrenia, which is acomplex, multifactorial clinical syndrome.

• The choice of a selected strain of animal to model a specific disease could be related to economicalaspects and not to scientific evidence.

• The indiscriminate use of strains could represent a potential source of conflicting results in scientificliterature.

Introduction

Early adverse experiences and especially vulnerabil-ity to these events may ‘shape’ a pre-existing geneticvulnerability to stress and disease. Thus, young ani-mals raised under adverse-rearing conditions, suchas social isolation or maternal separation, presentwith long-lasting behavioural and physiologicalchanges in adulthood that resemble depression and/orschizophrenia, including increased anxiety, compro-mised cognitive function, poor social interaction aswell as various depressive-like behaviours (1). Froma clinical point of view, depression and schizophre-nia lie on a continuum of symptom presentation andunderlying biology. Indeed, schizophrenic patientsoften show a depressive state as prodrome of thepsychiatric disorder (2). Rearing rats in social iso-lation from weaning results in long-term effectson brain structure, neurotransmitter function andbehaviour and many of the effects mimic thoseseen in schizophrenia and depression (3). Specifi-cally, isolation rearing of rats has been regardedas an ‘environmental model’ of schizophrenia (4)since the long-term alterations induced by thisnon-pharmacological method resemble symptomsseen in schizophrenic patients (5), such as hyper-reactivity to novel environments and cognitiveimpairment (6,7). Impairments in prepulse inhibition(PPI) are often reported in isolated rats (8) and inpatients with schizophrenia (9). However, this mayalso be observed in other psychiatric disorders suchas depression, where it may reflect stimulus overload-induced cognitive fragmentation. Moreover, loss ofsocial contact and behavioural withdrawal are asso-ciated with both schizophrenia and depression inhumans (10).

The isolation rearing model is based on theobservation that rats reared in single housing fromweaning throughout adulthood exhibit schizophrenia-and depression-like behavioural, morphological andneurochemical abnormalities (11). Among the mostcommon behavioural and morphological changesobserved in isolated rats are deficits in senso-rimotor gating, increased locomotor activity (12),anxiety-related behaviours (13) and decreased cor-tical and hippocampal synaptic plasticity (11). The

dopaminergic hypothesis is one of the most widelyaccepted theories regarding the aetiology of schizo-phrenic symptoms, such that impairment in thedopaminergic system could represent the neurochem-ical basis of these behavioural alterations (14). More-over, it also explains the common use of dopamine(DA) antagonists as clinically effective antipsychoticagents. Nonetheless, there is evidence that a keyeffect of social isolation may be loss of neuronalplasticity combined with change in the functionalityof various cortical and hippocampal neurotransmit-ters, including glutamate and serotonin (5-HT) (14).Although reduced glutamate function may under-lie the deficits in memory function, this can bereversed by administration of a 5-HT6 receptor antag-onist (15), emphasising the importance of cross-talkbetween these two transmitters. Moreover, manynew generation antipsychotics target 5-HT in theirpharmacological profile (14) and are effective notonly for schizophrenia but also for treatment-resistantdepression (16). However, there is a wealth of evi-dence showing that gender and age can affect boththe degree and direction of the resultant long-termchanges in behaviour and neurochemistry producedby exposure to early life adversity in the rat (17).

An additional variable in isolation experiments thathas not been extensively characterised is the extentto which the strain of rat used in the experimentsmay affect the results. Indeed social behaviour dif-fers by strain, making it a significant variable whenconsidering the influence of social-rearing conditionson later behaviour (18). Few published studies havecompared two different rat strains, such as Wistar(W) and Lister Hooded (LH), with respect to theirresponse to an adverse condition. However, thereis a surprising lack of comparative studies on LHand W rats of both genders, which motivated us touse these two strains. In particular, we have cho-sen the LH strain for our studies on isolation rear-ing as it has been found to produce consistent andreproducible behavioural effects with the isolationprocedure (15). Specifically, LH and W rats haverevealed considerable behavioural differences in kin-dling responses, anxiety (19), attention tasks, openfield and food-hoarding behaviour (20). The results

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of these studies seem to support increased anxietyand lower cognitive performance in W compared toLH rats.

The above reported scenario raises the intriguingquestion as to whether difference in these rat strainsmay have a neurochemical basis and whether a dif-ferent genetic background could be responsible forpossible neurochemical alterations in isolated rats(ISO). Thus, the first aim of this study was to evalu-ate the neurochemical profile of W and LH rat strainsby examining tissue content of DA, 5-HT and rela-tive metabolism, in several brain regions involvedin mental disorders, such as prefrontal cortex (PFC),nucleus accumbens (NAC), hippocampus (Hipp) andstriatum (St) of group housed (GRP) and ISO LHand W rats. Moreover, besides alterations in severalbrain areas, functional imaging and neuropsycholog-ical studies have provided convincing evidence forthe critical involvement of the PFC in schizophre-nia (21). Hence, in a second experiment, in vivomicrodialysis was performed in the PFC of W ratsas well as LH animals, either in GRP or ISO condi-tions to determine basal in vivo concentrations of theabove transmitters.

Materials and methods

Animals

The present study was conducted in W and LHadult male rats. They were housed at constantroom temperature (22 ± 1 ◦C) and relative humidity(55 ± 5%) under a 12-h light/dark cycle (lights onfrom 07:00 to 19:00 h). Food and water were freelyavailable.

Procedures involving animals and their care wereconducted in conformity with the institutional guide-lines that are in compliance with national (D. L. No.116, G. U., Suppl. 40, 18 Febbraio 1992, CircolareNo. 8, G. U., 14 Luglio 1994) and international lawsand policies (EEC Council Directive 86/609, OJ L358, 1, December 12, 1987; Guide for the Care andUse of Laboratory Animals, US National ResearchCouncil, 1996). All efforts were made to minimisethe number of animals used and their suffering.

Social isolation protocol

For the social isolation procedure (22), rats weremated such that all the mothers gave birth within 1week. At weaning (postnatal day 21), the pups wereseparated from their mothers and divided and rearedeither in ISO (one rat per cage) or in GRP (three orfour rats per cage) such that each litter contributedonly one subject to the GRP and one subject to theISO condition to avoid a litter effect (23). Animalswere disturbed only for cleaning purposes, which

consisted of changing the cage once a week for ISOand twice a week for GRP animals. ISO and GRPrats maintained visual, auditory and olfactory contactwith the other animals throughout the studies. Allexperiments were conducted at the end of 6–8 weeksof isolation rearing after body weight assessment.

Post-mortem tissue preparation

Rats were killed by decapitation and brains imme-diately removed. For dissection, the brains wereplaced dorsal side up in an ice-chilled rat brainmatrix (World Precision Instruments Inc., Sarasota,FL, USA) with slits spaced at 1 mm using an ice-chilled razor blade, brain slices containing the tar-get regions were dissected according to the atlasof Paxinos and Watson (24) (Fig. 1). Then PFC,NAC, St and Hipp were collected by tissue dissec-tion and immediately frozen on dry ice. For the bio-genic amine extraction, samples were homogenisedin 10 volumes (w/v) of perchloric acid 0.1 M. Thehomogenates were stored on ice for 30 min and thencentrifuged at 10000 × g for 10 min at 4 ◦C. Thesupernatants were then filtered (0.45 μm syringe fil-ters, Millipore, Vimodrone, Milan, Italy) and diluted(PFC and NAC not diluted, St 1:4, Hipp 1:2) with0.1 M perchloric acid before high-performance liquidchromatography (HPLC) analysis. DA turnover wasexpressed as (HVA+DOPAC)/DA and 5-HT turnoveras 5-HIAA/5-HT.

In vivo microdialysis

Rats were anaesthetised with 3.6 ml/kg Equithesinintraperitoneally (1.2 g sodium pentobarbital; 5.3 gchloral hydrate; 2.7 g MgSO4; 49.5 ml propyleneglycol; 12.5 ml ethanol and 58 ml distilled water)and secured in a stereotaxic frame (David KopfInstruments, Tujunga, CA, USA). A hole was drilledin the frontal bone and a small incision was made inthe dura with a bent needle tip. The probe (4 mm)was lowered slowly into the PFC (AP = +3.7, ML =+0.7, DV = −4.8) from bregma according to thePaxinos and Watson atlas (24). The dialysis probes(AN69 Hospal SpA,; 20 kDa cut-off) were preparedas described by Robinson and Whishaw (25). Therats were allowed to recover from anaesthesia forat least 15 h before the neurotransmitter releasestudy (26).

On the day of the experiment, the fibres wereperfused with an artificial cerebrospinal fluid con-taining: 145 mM NaCl, 3 mM KCl, 1.26 mM CaCl2,1 mM MgCl2 and 1.4 mM Na2HPO4 in distilledwater (1 l); the solution was buffered at pH 7.3with a 0.6 mM NaH2PO4 and filtered (0.45 μm).The fibres were perfused at a constant flow rate

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(a) (b)

(c) (d)

Fig. 1. Regions of the post-mortem tissue preparation. The locations of the razor blades used to cut the adjacent coronal slices areindicated in a sagittal view of the rat brain with the ventral surface down. Brain regions of interest were dissected on the superiorface of the coronal slices as indicated (a: PFC; b: Hipp; c: NAC; d: St).

of 2 μl/min using a CMA/100 microinjection pump(CMA Microdialysis, Stockholm, Sweden). Themicrodialysis membrane was allowed to stabilisefor 2 h. Then, samples were collected at 20-minintervals. All samples of perfusate were immediatelyanalysed by HPLC.

The position of the microdialysis probe was ver-ified by histological procedures at the end of eachexperiment. DA and 5-HT concentrations were deter-mined by HPLC coupled with an electrochemicaldetector (INTRO, Antec Leyden, The Netherlands).Amines separation was performed by an LC18reversed phase cartridge column (15 cm × 2 mm,3 μm; Phenomenex, Castel Maggiore, Bologna,Italy). The detection was accomplished by a Uni-jet cell (BASi) with a 6-mm diameter glassy car-bon electrode at a working potential of 0.65 V ver-sus Ag/AgCl. The mobile phase used was 85 mMCH3COONa, 0.8 mM octan sulfonic acid, 0.3 mMethylenediaminetetraacetic acid, 15 mM NaCl, meth-anol 6%, solved in distilled water, buffered at pH

4.85 with CH3COOH and filtered. The flow rate usedwas 0.220 ml/min.

Statistical analysis

For the post-mortem tissues analysis, content ofneurotransmitters were analysed by two-way analysisof variance (ANOVA), with main factors of housingcondition (r), comparing ISO and GRP rats, andstrain (st), comparing W and LH animals.

For the microdialysis analysis, four consecutivesamples were collected (80 min). Since differencesbetween rearing conditions and strain were time inde-pendent, individual comparisons between marginalmean values of neurotransmitters levels (pooled dataof 4 samples for each factor) were made and anal-ysed by ANOVA using housing condition and strainas between-subjects factors.

In the case of significant main effects, thedifferences between individual means were assessedwith the post hoc Tukey’s test. Differences wereconsidered significant only when p-values were lessthan 0.05.

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Results

Body weight

Animal body weight was evaluated at the end ofthe isolation-rearing period. This parameter wasestimated in order to consider any possible grosssign of animal distress that could relate to thedifference in brain neurochemistry, leading to datamisinterpretation.

After 6–8 weeks of rearing, no significant differ-ence in the body weight (GRP LH: 338.7 ± 8.7 g,ISO LH: 354 ± 7.3 g, GRP W: 339.4 ± 10.2 g, ISOW: 320.5 ± 21.7 g) of the two different rat strains[F (st)1,26 = 1.203, n.s.] or the two different rear-ing conditions [F (r)1,26 = 0.0121, n.s.] was observed(data not shown). Moreover, no interaction betweenrearing conditions and strain was found [F (r ×st)1,26 = 1.312, n.s.].

Post-mortem analysis

In order to assess the neurochemical profile of W andLH rat strains, we examined tissue content of DA, 5-HT and metabolites in several brain regions involvedin mental disorders, including the PFC, NAC, Hippand St from GRP and ISO rats from both strains.

Prefrontal cortex

A significant difference in DA content between Wand LH rats, with more DA in the PFC of LHthan W rats, was observed (Fig. 2a). Moreover, inLH strain, ISO rats showed higher DA contentthan GRP animals [F (st)1,23 = 49.070, p < 0.001;F (r)1,23 = 2.743, n.s.; F (r × st)1,23 = 7.173, p <

0.05] (Fig. 2a). Moreover, DA turnover in LH rats

(a) (b)

Fig. 2. Effects of social isolation on PFC content ofmonoamines in W (black bar) and LH (grey bar) rats. ∗∗p <0.01, ∗∗∗p < 0.001 comparing W and LH rats in the same rear-ing condition. ##p < 0.01 comparing GRP and ISO rats of thesame strain. All values are the mean ± SEM (n = 5–8 rats).

was significantly lower compared to W, indepen-dently from rearing conditions (two-way ANOVA,p < 0.01, Table 1), while no difference was evidentin W rats after isolation.

W and LH differed significantly in 5-HT content,being 5-HT content higher in LH rats, irrespective ofhousing conditions [F (st)1,22 = 28.061, p < 0.001;F (r)1,22 = 1.261, n.s.; F (r × st)1,22 = 1.154, n.s.](Fig. 2b). In addition, isolation caused an increase in5-HT turnover only in W strain (two-way ANOVA,p < 0.05, Table 1).

Hippocampus

W and LH rats differed significantly in the contentof DA [F (st)1,24 = 37.746, p < 0.001; F (r)1,24 =0.327, n.s.; F (r × st)1,24 = 2.720, n.s.], either inGRP or in ISO rats, showing higher DA contentin LH rats (Fig. 3a). In regard to DA metabolism,LH strain showed a DA turnover significantlyhigher (two-way ANOVA, p < 0.05), however inthis strain such parameter was increased by isolation(two-way ANOVA, p < 0.001) (Table 2). W ratsexhibited higher 5-HT content only in GRP group.

Table 1. Effects of social isolation on tissue DA and 5-HT turnover in PFC of W andLH rats

PFC (HVA+DOPAC)/DA 5-HIAA/5-HT

W GRP 2.353 ± 0.61 9.399 ± 3.13W ISO 3.263 ± 0.62 15.91 ± 3.41†

LH GRP 0.139 ± 0.08∗ 11.116 ± 2.9LH ISO 0.048 ± 0.01∗ 12.517 ± 1.0

All values are the mean ± SEM (n = 5–8 rats).∗p < 0.001 vs. W rats in the same rearing condition.†p < 0.05 vs. GRP rats of the same strain.

(a) (b)

Fig. 3. Effects of social isolation on Hipp content ofmonoamines in W (black bar) and LH (grey bar) rats. ∗∗p <0.01, ∗∗∗p < 0.001 comparing W and LH rats in the same rear-ing condition. #p < 0.05 comparing GRP and ISO rats of thesame strain. All values are the mean ± SEM (n = 5–8 rats).

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Table 2. Effects of social isolation on tissue DA and 5-HT turnover in Hipp of W andLH rats

HIPP (HVA+DOPAC)/DA 5-HIAA/5-HT

W GRP 1.16 ± 0.12 2.40 ± 0.26W ISO 1.48 ± 0.16 3.05 ± 0.47LH GRP 0.40 ± 0.07∗ 4.41 ± 0.23∗

LH ISO 0.88 ± 0.14†,‡ 0.20 ± 0.02∗

All values are the mean ± SEM (n = 5–8 rats).∗p < 0.001 vs. W rats in the same rearing condition.†p < 0.05 vs. W rats in the same rearing condition.‡p < 0.05 vs. GRP rats of the same strain.

Moreover, social isolation significantly reduced 5-HTlevels in W strain [F (st)1,23 = 15.665; p < 0.001;F (r)1,23 = 2.309, n.s.; F (r × st)1,23 = 3.904, p <

0.05] (Fig. 3b). No difference in turnover was foundin W, while in LH the isolation rearing significantlyreduced, 5-HT metabolism (two-way ANOVA, p <

0.0001, Table 2).

Nucleus accumbens

As shown in Fig. 4a, a significant effect of strain wasevident [F (st)1,23 = 72.062, p < 0.001; F (r)1,23 =0.324, n.s.; F (r × st)1,23 = 0.160, n.s.] reflectingan increased DA content in LH rats in comparisonwith W animals, either in GRP or ISO animals.Moreover, DA turnover in LH rats was significantlylower compared with W, independently from rearingconditions (two-way ANOVA, p < 0.001, Table 3),while no difference was evident in W rats afterisolation.

A strain-dependent effect was observed also whenconsidering 5-HT content, either in GRP or in ISO

(a) (b)

Fig. 4. Effects of social isolation on NAC content ofmonoamines in W (black bar) and LH (grey bar) rats. ∗p <0.05, ∗∗∗p < 0.001 comparing W and LH rats in the same rear-ing condition. ##p < 0.01 comparing GRP and ISO rats of thesame strain. All values are the mean ± SEM (n = 5–8 rats).

Table 3. Effects of social isolation on tissue DA and 5-HT turnover in NAC of W andLH rats

NAC (HVA+DOPAC)/DA 5-HIAA/5-HT

W GRP 20.79 ± 3.63 4.81 ± 0.39W ISO 28.67 ± 3.04 9.03 ± 1.55†

LH GRP 0.52 ± 0.13∗ 0.23 ± 0.06∗

LH ISO 0.7 ± 0.09∗ 0.26 ± 0.04∗

All values are the mean ± SEM (n = 5–8 rats).∗p < 0.01 vs. W rats in the same rearing condition.†p < 0.05 vs. GRP rats of the same strain.

(a) (b)

Fig. 5. Effects of social isolation on St content of monoaminesin W (black bar) and LH (grey bar) rats. ∗p < 0.05,∗∗p < 0.01, ∗∗∗p < 0.001 comparing W and LH rats in thesame rearing condition. All values are the mean ± SEM(n = 5–8 rats).

rats. Indeed, LH animals showed decreased neuro-transmitter levels. Moreover, the analysis revealeda significant decreased 5-HT content only in Wrats, consequent to isolation rearing [F (st)1,23 =25.091; p < 0.001; F (r)1,23 = 9.647, p < 0.01; F (r× st)1,23 = 5.200, p < 0.05] (Fig. 4b). Finally, iso-lation caused an increase in 5-HT turnover in Wstrain (two-way ANOVA, p < 0.05 Table 3); how-ever, independently from rearing conditions, LH ratsshowed lower 5-HT turnover (two-way ANOVA,p < 0.05, Table 3).

Striatum

Irrespective of housing conditions, the analysisshowed a significant increase in DA content in LHrats with respect to W strain [F (st)1,21 = 9.922,p < 0.01; F (r)1,21 = 0.00715, n.s.; F (r × st)1,21 =0.0588, n.s.] (Fig. 5a), while no difference wasevident in DA turnover (Table 4). As far as 5-HT, a strain-dependent effect was evident eitherin GRP or ISO rats, resulting in increased 5-HTcontent in LH animals [F (st)1,21 = 37.693, p <

0.001; F (r)1,21 = 1.034, n.s.; F (r × st)1,21 = 0.818,

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Table 4. Effects of social isolation on tissue DA and 5-HT turnover in St of Wand LH rats

St (HVA+DOPAC)/DA 5-HIAA/5-HT

W GRP 0.13 ± 0.01 0.33 ± 0.07W ISO 0.12 ± 0.01 0.6 ± 0.08∗

LH GRP 0.16 ± 0.02 0.17 ± 0.01†

LH ISO 0.22 ± 0.03 0.17 ± 0.02‡

All values are the mean ± SEM (n = 5–8 rats).∗p < 0.05 vs. GRP rats of the same strain.†p < 0.05 vs. W rats in the same rearing condition.‡p < 0.001 vs. W rats in the same rearing condition.

n.s.] (Fig. 5b). In addition, isolation caused anincrease in 5-HT turnover in W strain (two-wayANOVA, p < 0.05, Table 4). In LH strain 5-HTturnover was lower compared to W, independentlyfrom rearing conditions (two-way ANOVA, p <

0.05, Table 4). In addition, isolation caused anincrease in 5-HT turnover only in W strain (two-wayANOVA, p < 0.001, Table 4).

Overall differences in post-mortem tissue content ofneurotransmitters

Figure 6 summarises the overall differences inpost-mortem tissue content of the neurotransmit-ters considered in this study (Fig. 6a: DA; Fig. 6b:5-HT). Results showed the different strain-dependentresponses of ISO rats in percent relative to normalGRP animals.

In vivo microdialysis

To understand if social isolation alters neurotrans-mitter release, we have performed microdialysis in

freely moving animals to measure the extracellularconcentration of DA and 5-HT in PFC of rats.

Since differences between rearing conditions andstrain were time independent, individual comparisonsbetween marginal mean values (pooled data of foursamples for each factor) were made and a two-wayANOVA was performed.

Results showed higher DA basal levels in LHrats compared with W rats, irrespective of housingconditions [F (st)1,91 = 45.96, p < 0.001; F (r)1,91 =0.753, n.s.; F (r × st)1,91 = 2.104, n.s.] (Fig. 7a).

Basal levels of 5-HT were higher in W withrespect to LH rats, but only in GRP conditions. More-over, social isolation decreased 5-HT concentra-tions only in W rats [F (st)1,91 = 21.513, p < 0.001;

(a) (b)

Fig. 7. Effects of social isolation on in vivo PFC levels ofmonoamines in W (black bar) and LH (grey bar) rats. ∗∗∗p <0.001 comparing W and LH rats in the same rearing condition.###p < 0.001 comparing GRP and ISO rats of the same strain.All values are the mean ± SEM (n = 5–8 rats).

(a) (b)

Fig. 6. The different strain-dependent responses to ISO rats in percent relative to normal GRP animals for DA and 5-HT in PFC,Hipp, NAC and St of W ( ) and LH ( ) rats.

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F (r)1,91 = 6.546, p < 0.05; F (r × st)1,91 = 8.384,p < 0.01] (Fig. 7b).

Discussion

This study suggests that the choice of rat strain canmake a crucial difference when analysing the effectsof isolation rearing on regional brain monoaminecontent. Clear and distinct neurochemical profilesin the brain areas examined were observed. In thisregard, no extraneous experimental variables, such asbody weight, could be forwarded as an explanation,as no significant difference in the body weight of thetwo different rat strains was observed.

Often, the rationale behind selecting one strain canbe mainly speculative and affected by anthropomor-phic inference (27), although it can be addressed byeconomical evaluations of the experiments and by thesurprising lack of references in comparative studieson behavioural and monoaminergic alterations fol-lowing social isolation rearing in W and LH rats, asshown in the literature review by Lukkes and co-workers (13).

Thus, the discussion of these results shouldconsider two aspects: (a) strain-dependent differ-ences in neurochemistry and (b) strain-related differ-ences consequent to isolation. Furthermore, a fun-damentally important question should be addressed:could strain-dependent differences explain differen-tial effects of isolation, and as such be an importantconsideration for using the model in basic studies ofschizophrenia or depression?

When analysing strain-dependent neurochemicaldifferences, it appeared that brain regions in LH rats,at least those examined, contained higher DA levelswith respect to similar brain areas in W rats. In line,in this strain DA metabolism resulted reduced withthe exception of St.

Thus, considering that the dopaminergic hypoth-esis is one of the most widely accepted hypothe-ses regarding the aetiology of schizophrenic symp-toms (28), these results seem to explain at leastsome of the behavioural contrasting results found inthe isolation-based animal model of schizophrenia,where studies are performed in different rat strains,and why LH rats are a preferred strain.

In this regard, the PPI paradigm used in rodentshas been proposed to model the attention impair-ments and deficits in psychomotor gating seen in psy-chiatric disorders such as schizophrenia (29). Thereis converging evidence for the important involve-ment of dopaminergic systems in the control ofPPI. Specifically, neurotoxic lesion experiments (30)support the notion that increased DA transmissionand DA receptor super- or sub-sensitivity under-lie the disruption in PPI consequent to the above

interventions. Indeed, it has been previously reportedthat LH rats demonstrate a greater reduction in startlereactivity relative to W animals (31).

Interestingly, the lower basal DA levels found inall brain areas examined in W rats with respect toLH animals could be consistent with the findingthat the W rat strain appears to be more resistantto developing certain behaviours typical of animalssubjected to ISO, specifically disruption of PPI.Indeed, raised accumbal DA, as it was particularlyevident in LH rats, will imply an increase in salienceculminating in an attenuation in sensorimotor gatingand an associated decrease in startle reactivity, whilereduced DA in W rats imply reduced salience andan increase in startle reactivity. These data seem tosuggest that optimum levels of DA are necessary toobserve schizophrenic-like behavioural alterations inspecific rat strains (32).

Moreover, it has been demonstrated that LH ratsdisplay increased locomotor activity in comparisonwith W rats (31). The higher DA content found in theSt of LH rats with respect to W rats could accountfor this behavioural evidence.

Strain-related differences were also observed inthe serotonergic system. In particular, in vivo resultsshowed decreased extracellular 5-HT concentrationsin the PFC of LH rats, with respect to W animals.Prefrontal DA is under strong restraint via corticalserotonergic projections (33), such that reduced 5-HT may underlie the marked elevations in PFC DAobserved in LH rats. Surprisingly, however, post-mortem tissue content of 5-HT were increased inthe PFC and St of LH rats, while the same ratsshowed lower 5-HT content in the NAC and Hipp.Additionally, in these areas 5-HT metabolism wasreduced compared to W rats, independently fromisolation-rearing condition. In this strain, however,hippocampal 5-HT turnover was increased by isola-tion. The discrepancy between in vivo and ex vivoresults found in the PFC may depend on the loss ofneuronal connection in post-mortem analyses, as wellas reflecting the whole neuronal pool of transmitterversus the synaptic levels.

The different effect found in microdialysis studywith respect to ex vivo data may be due to thefact that chronic stress of isolation decreased 5-HTrelease (11), thus increasing tissue content.

Rearing rats in isolation produced specific strain-dependent changes in the post-mortem tissue con-centrations and in vivo basal levels of monoamines.In particular, LH-isolated animals had significantlyhigher concentrations of DA in the PFC whencompared to GRP LH controls, but no alterationswere observed in the NAC, St or Hipp. With regardto W rats, no variations of DA concentrations werefound after isolation in all brain areas analysed. This

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re-emphasises the critical role of PFC DA in mediat-ing the behavioural responses akin to schizophrenia,and the more limited role for DA in the latter regions.

Selective alterations in cortical DA function havebeen reported previously following isolation in LHrats (11). As in this study, the effects on DA in thePFC were also specific, as no alterations in the NACor St were found in the ISO LH rats.

The impact of social deprivation in rats is,however, not restricted to effects on dopaminer-gic transmission. In this study, post-mortem studiesrevealed that 5-HT content was unaffected in LHrats by rearing conditions in all brain areas con-sidered. Conversely, 5-HT content was significantlydecreased in the NAC and Hipp of W rats fol-lowing isolation. It could be hypothesised that thedecreased 5-HT levels in these brain regions follow-ing chronic isolation in W rats cannot be compen-sated by an increase in 5-HT synthesis, leading thento depressive-like behaviour. Indeed, reduced 5-HTcontent or 5-HT release has also been found in cortexand Hipp of ISO animals (34). Turning to the in vivodata, 5-HT was markedly reduced in the PFC in Wrats following isolation, but no effects were observedin LH rats. Serotonergic changes subsequent to isola-tion are consistent with data describing the ability ofsome antidepressant compounds to reverse the effectsof isolation rearing (35).

Then, it is pertinent to note that neuropsychi-atric disorders like depression have been considereda consequence of inadequate neurochemical adap-tation in response to stressors (36). Interestingly inthis context, it must be underlined that depressionis a common symptom before (37), during and afteran episode of schizophrenia (38). These results seemto support the developmental hypothesis that earlyadverse environmental life events could predisposeto adult depressive-like response (39). However, fur-ther studies should be conducted to evaluate theinvolvement of other neurotransmitters. In particu-lar, it is hypothesised that early life stressful eventsmay lead to glutamate hyperfunction, thus prevent-ing neuronal connections to the cortex and resultingin dopaminergic and serotonergic dysfunctions (40).In line with this, further investigations directed toassess glutamate involvement in different rat strainsare warranted.

In conclusion, this data provide the first evidencethat choice of strain and genetic background are ofimportance when considering an environmental ani-mal model of schizophrenic disorders. From theseresults we could hypothesize that the neurochemicalphenotype of the LH rat, particularly with respectto DA/5-HT changes in the PFC, may constitutea valuable genetic model of vulnerability for thedevelopment of schizophrenic manifestations in ISO

animals, while the W rat strain could better pro-vide information on several parameters associatedwith depression or in schizophrenia comorbid withdepression.

Acknowledgements

G. W. serves as Editor-in-Chief for Acta Neuropsychiatrica,but was not involved in and actively withdrew from thereview/decision process of this paper. No other conflicts ofinterest are to be declared.

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