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Short Communication

Treatment with olanzapine increases cell proliferation in thesubventricular zone and prefrontal cortex

William Green, Parag Patil, Charles A. Marsden, Geoffery W. Bennett, Peter M. Wigmore⁎

Institute of Neuroscience, School of Biomedical Sciences, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK

A R T I C L E I N F O

⁎ Corresponding author. Fax: +44 115 9709 25E-mail address: peter.wigmore@nottingha

0006-8993/$ – see front matter © 2005 Elsevidoi:10.1016/j.brainres.2005.11.047

A B S T R A C T

Article history:Accepted 2 November 2005Available online 6 January 2006

The present study examines the effect of chronic treatment with two atypical neuroleptics,commonly used to treat schizophrenia. Adult rats were given either risperidone orolanzapine in their drinking water for 21 days. Memory was assessed on the first and lastday of treatment using an object discrimination test, and the rate of cell proliferation in thesubventricular zone (SVZ), dentate gyrus (DG) and prefrontal cortex (PFC) was quantified byimmuno staining for Ki-67. The results show that both risperidone and olanzapinesignificantly improved performance in object discrimination after 21 days, andadditionally, olanzapine significantly increased cell proliferation in the SVZ and PFC butnot the DG.

© 2005 Elsevier B.V. All rights reserved.

Keywords:NeurolepticNeurogenesisCognitionSchizophreniaAdult brain

The continued proliferation of precursor cells which cangenerate new neurones in the adult brain has attractedincreasing attention and is now thought to occur in the brainsof all mammals, including man (for review, see Gould andGross, 2002). Although many regions of the postnatal braincontain progenitor cells, capable of generating both neuronesand glia, in vivo neurogenesis is restricted to a small numberof specific regions. The largest of these is the subventricularzone (SVZ) of the lateral walls of the lateral ventricles (Brazelet al., 2003). Cells produced here have been shown, in rodents,to pass via the rostral migratory stream, to the olfactory bulbswhere they differentiate into olfactory bulb granule andperiglomerular neurones. Recent evidence now shows thatthe SVZ may also contribute new neurones to other brainregions (amygdala, cortex and striatum) in adult animals(Bernier et al., 2002; Dayer et al., 2005). The expression of theproteoglycan NG2 has been shown to be a marker forproliferating cells which can differentiate into neurones, andNG2+ cells are found both in the SVZ and in the cortex, raisingthe possibility that new neurones may be generated in situ at

9.m.ac.uk (P.M. Wigmore).

er B.V. All rights reserved

the sites in which they will integrate (Dayer et al., 2005;Belachew et al., 2003). However, most evidence indicates thatthe low levels of cell proliferation which occur within thecerebral cortex, under non-pathological conditions, generatecells with a glial phenotype (Levison et al., 1999; Magavi et al.,2000). In agreement with this, recent studies have shown thattreatment of animals with electroconvulsive seizuresincreases the numbers of proliferating cells in the frontalcortex, but that these differentiate into glia or endothelial cells(Madsen et al., 2005; Ongur and Heckers, 2004).

Neurogenesis also takes place in the hippocampus, in thesubgranular zone (SGZ), adjacent to the dentate gyrus (Gouldet al., 2000; Cameron and McKay, 2001). Cells generated in theSGZ migrate into the dentate gyrus before differentiating intoneurones and making functional connections.

The study of cell proliferation in the adult brain has beenstimulated by observations that the rate of cell division isaffected by external influences and can be modulated by drugtreatment. Interest has focused on the reduction in hippocam-pal neurogenesis, associated with depression and stress, and

.

Fig. 1 – Section through the dentate gyrus (star) stained withpropidium iodide (red) to show all nuclei and Ki-67 (green).Ki-67 positive cells are seen in the subgranular zone adjacentto the dentate gyrus.

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its increase with chronic antidepressant treatment (Brown etal., 2003; Duman et al., 2001). The response to antidepressantsrequires the generation of new neurones (Santarelli et al.,2003), and it is possible that increases in cell proliferation arealso associatedwith functional changes in other brain regions.

Chronic administration of atypical neuroleptics has beenreported recently to increase cell proliferation in the SVZ(Wakade et al., 2002) and cortex and hippocampus (Kodama etal., 2004). Schizophrenia is associated with a cognitiveimpairment and decreased neuronal function in the prefrontalcortex (PFC), (Friedman et al., 1999). Atypical neuroleptics arereported to improve cognition in schizophrenics, suggestingthat these drugs may affect the hippocampus and PFC,both key areas in memory and cognition (Green et al.,1997; Nieoullon, 2002). To further elucidate the action ofatypical neuroleptics, we have examined the effect ofchronic administration of olanzapine and risperidone oncognitive behavior and cell proliferation in the SVZ of thelateral ventricle, the SGZ of the hippocampus and the PFC.

The object discrimination test was carried out on each ofthe control, risperidone and olanzapine treated groups (n = 6)at days 1 and 21 of the drug regimen. On day 1 (prior to drugtreatment), there was no significant difference in discrimina-tion scores between groups (data not shown).

After 21 days of drug treatment, animals treatedwith eitherrisperidone or olanzapine showed an increase in the timespent examining the novel object such that there was asignificant increase in mean discrimination scores, comparedto controls (Table 1).

Ki-67 staining was carried out on 4 20-μm-thick brainsections per rat, for each of the prefrontal cortex, dentate gyrusand subventricular zones. All sections were 200 μmapart. Thisresulted in a sample size of 24 sections per treatment group foreach area of the brain examined. Ki-67 positive cells wereeasily identified, occurring as either single cells or clusters of 2–6 cells along the inner edge of the dentate gyrus (Fig. 1) or SVZ.In the PFC, cells occurred either singly or as pairs of Ki-67positive cells.

In the prefrontal cortex, there was no significant change inthe number of Ki-67 positive cells following risperidone

Table 1 – Results of the object discrimination test andmean numbers of Ki-67 positive cells per section

Discriminationration

Mean no.of Ki-67positivecells persectionof PFC

Meanno. ofKi-67

positivecells persection

ofdentategyrus

Mean no.of Ki-67positivecells persectionof SVZ

Control 0.491 (0.085) 8.5 (0.99) 8.0 (1.03) 9.0 (2.18)Risperidone

treated0.714 (0.052)* 9.8 (1.08) 10.0 (0.97) 10.3 (0.99)

Olanzapinetreated

0.778 (0.026)* 13.0 (1.53)* 10.8 (0.98) 16.2 (0.96)*

Figures in brackets indicate standard error of the mean, asteriskindicates significant difference from the controls.

treatment, compared to controls. In contrast, there was asignificant increase (44%) in the number of Ki-67 positive cellsin the PFC of olanzapine treated rats, compared to controls(Table 1).

Similarly in the subventricular zone, risperidone had nosignificant effect on the number of Ki-67 positive cells butolanzapine produced a significant increase in the number ofpositive cells in this region (Table 1).

Finally in the dentate gyrus of the hippocampus, although asmall increase in Ki-67 positive cells was seen with drugtreatment, neither risperidone nor olanzapine had a signifi-cant effect on the number of Ki-67 positive cells (Table 1).

The results show that both olanzapine and risperidonesignificantly improve object discrimination. Olanzapine sig-nificantly increased cell proliferation in the prefrontal cortexand subventricular zone. Cognitive impairment, which isconsidered to underlie schizophrenia, is linked to abnormal-ities within the PFC, and severe deficits in working memoryare commonly observed in schizophrenic patients (Friedmanet al., 1999; Nieoullon, 2002). For this reason, a measure ofworking memory is useful in assessing the action of neuro-leptics. In the present study, treatment with two atypicalantipsychotics, risperidone and olanzapine significantly im-proved working memory as indicated by the increaseddiscrimination ratios in the object discrimination test.

Olanzapine has been previously reported to increase cellproliferation in the rat SVZ by some authors (Wakade et al.,2002) but not by others (Wang et al., 2004; Kodama et al.,2004). The present study found a significant increase in cellproliferation in this region after olanzapine treatment butnot with risperidone. Under normal conditions, cells gener-ated in the rodent SVZ, migrate rostrally and differentiate asneurones or supporting cells within the olfactory bulbs (Loisand Alvarez-Buylla, 1994; Lois et al., 1996; Luskin, 1993). Inman, a similar proliferation of stem cells occurs in the lateralventricle SVZ, and newly generated neurones are found inthe olfactory bulbs (Sanai et al., 2004; Bedard and Parent,

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2004). Patients suffering from schizophrenia have reducedolfactory bulb and medial temporal lobe volumes which arein turn associated with impaired olfaction (Purdon and Flor-Henry, 2000; Turetsky et al., 2000; Moberg and Turetsky, 2003;Turetsky et al., 2003). These anatomical and sensory deficitshave been correlated with an increased proportion ofimmature neurones in the olfactory bulbs of schizophrenicpatients and has led to the suggestion that a failure in theprocesses of neurodevelopment underlies aspects of thisdisease (Arnold et al., 2001). It is therefore of interest to notethat olanzapine inhibits apoptosis and increases the prolif-eration of neural cells in vitro and stimulates nerve growthfactor synthesis in vivo (Li et al., 1999; Dwyer et al., 2003;Parikh et al., 2004). It is however unclear what impactincreased proliferation within the SVZ would have on eitherolfaction or the function of regions of cortex to whicholfactory neurones project.

A small but non-significant increase in cell prolifera-tion in the present study was seen in the dentate gyrusof the hippocampus after treatment with risperidone andolanzapine, a result similar to that reported previously byWakade et al. (2002). Other authors however have foundsignificant increases in proliferation in this region withsimilar or higher doses of olanzapine (Wang et al., 2004;Kodama et al., 2004), suggesting that proliferating cells inthis region respond to both atypical neuroleptics andantidepressants.

The present study also found a significant increase in thenumbers of dividing cells within the PFC after olanzapine butnot risperidone treatment. Under normal physiological con-ditions, a small number of dividing cells can be found in thisregion which continue to generate astrocytes and oligoden-drocytes throughout life (Levison et al., 1999; Magavi et al.,2000). Antidepressant drugs, electroconvulsive therapy andatypical antipsychotics all appear to act to increase prolifer-ation in this region (Wang et al., 2004; Kodama et al., 2004;Madsen et al., 2005). When the differentiation of the newlygenerated cells has been followed, the results have shown thatall new cells are non-neural (Kodama et al., 2004; Madsen etal., 2005). Changes in the PFC are of particular interest asalterations in this region are thought to underlie the cognitivedeficits seen in schizophrenia. Patients suffering from schizo-phrenia have a reduced thickness of prefrontal cortex due to areduced number of glia rather than changes in neuronaldensity (Sanfilipo et al., 2000; Cotter et al., 2001; Hof et al.,2003). This has led to the suggestion that abnormalities in glialcell function in this region may underlie some aspects of thisdisease. The finding in the present study that atypicalneuroleptics significantly increase numbers of dividing cellsin the PFC is in line with a report that prolonged antipsychotictreatment of primates increases the density of glia in the PFC(Selemon et al., 1999).

In summary, anti-psychotics in the present studyimproved cognitive function and in the case of olanzapine,this was associated with increased proliferation in the PFCand SVZ.

This study seeks to correlate the effects of antipsychoticdrug administration on cognitive behavior and cell prolifera-tion in different brain regions. Control (vehicle only) olanza-pine and risperidone treated groups were randomly selected

from the same group of animals, and behavioral testing andhistology were carried out on all animals.

Adult male hooded Lister rats were supplied and housed inthe Biomedical Services Unit, Queen's Medical Centre, Uni-versity of Nottingham. The treatment of animals was inaccordance with the Home Office guidelines on the use ofanimals for scientific research.

Animals were housed 3 to a cage with a 12-h light/darkcycle and randomly assigned to Control (n = 6), risperidone(kindly donated by Janssen Pharmaceuticals, n = 6) andolanzapine (Eli Lilly Pharmaceuticals [n = 6]) treated groups.On day 1 of the experiment (prior to drug treatment), an objectdiscrimination test was carried out on all animals as describedbelow. All rats were allowed ad libitum access to food andprovided with drug dissolved in drinking water.

The fluid intake of each cage of rats was assessed bymeasuring the weight of the water bottles daily. Individualrats were estimated to drink ∼28 ml in a 24-h period, andthe final concentration of each drug supplied in the drinkingwater was determined for each cage from the mean volumesconsumed and mean bodyweight of the rats. Risperidonewas administered at 0.5 mg/kg/day and olanzapine at 2 mg/kg/day. These doses were chosen to correspond to recom-mended human doses (BNF 2002) and are identical to thoseused in previous studies (Gao et al., 1998; Wakade et al.,2002). Plasma concentrations of olanzapine administered atthis dose by the same route have previously been measuredand shown to be 9.3 ng/ml (Gao et al., 1998).

The drug solutions were replaced every 3 days, and therewas no difference in water intake between drug-adminis-tered and control groups. Rats were weighed at the start ofthe experiment and after each week, and there was nosignificant difference in weight between groups at any time.On day 21, the object discrimination test was repeated asdescribed below.

Themethodwasmodified from that described by Ennaceurand Meliani (1992) and Wasuntarawat (2001). The objectdiscrimination test is an assessment of working memory,with no conditioning components and reference memoryaspects (Ennaceur and Meliani, 1992).

The behavior was recorded in clear PVC boxes(38.5 × 23.5 × 30 cm). The objects used in the tests were smallplastic bottles, water-filled to ensure stability and decoratedwith colored tape to create individual visually distinct designs.Between tests, the equipmentwas cleanedwith 10%ethanol toeliminate any odor cues. Rats were habituated to the testingbox on the day prior to testing. After being exposed to identicalbottles for 3 min, the animals experienced a 1-min retentiondelay before exposure to a both a familiar andnovel object for 3min. Object exploration was defined as directing the nose tothe object at less than 2 cm. The discrimination ratio (DR)describes the difference in time spent exploring a novel andfamiliar object, divided by the total time spent exploringobjects.

In order to check whether drug administration had ageneral effect on exploratory behavior or locomotion, the totaltime spent exploring both novel and familiar objects wascompared between control and drug treated animals. Nosignificant difference was found between groups using a one-way ANOVA.

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Following the object discrimination test on day 21, all therats were killed by rapid stunning followed by decapitation.The brains were removed and individual hemispheres placedin 30% sucrose on ice for 30 min before freezing inisopentane in liquid nitrogen. 20-μm cryostat (Microm)sections were cut and mounted on Apes coated slides. 5coronal brain sections were collected every 400 μm andBromomethyl blue staining was used to identify the PFCimmediately rostral to the hippocampus, the dentate gyrusand the subventricular zone. For each hemisphere, 4consecutive sections (each 400 μm apart) were selectedusing a systematic random sampling procedure (Mayhewand Burton, 1988) to assess cell proliferation in each of theregions (PFC, SVZ and SGZ).

Cell proliferation was assayed by immunodetection of theKi-67 protein, a marker of all stages of the cell cycle (Kee etal., 2002; Scholzen and Gerdes, 2000). Slides were immunos-tained for Ki-67 using a monoclonal antibody (Novocastra,UK) following the manufactures protocol. Sections werecounter stained with propidium iodide (Sigma) to show allcell nuclei. The number of Ki-67 positive cells in the specificareas of interest from each section was recorded by thesame investigator.

Data analysis used Wilcoxon signed-rank test or one-wayanalysis of variance (ANOVA) with post hoc Kruskal–Wallis.

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