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Treatment with olanzapine increases cell proliferation in the subventricular zone and prefrontal cortex
BR A I N R ES E A RC H 1 0 7 0 ( 2 00 6 ) 2 4 2 –24 5
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Short Communication
Treatment with olanzapine increases cell proliferation in the subventricular 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 LE I N FO
AB S T R A C T
Article history:
The present study examines the effect of chronic treatment with two atypical neuroleptics,
Accepted 2 November 2005
commonly used to treat schizophrenia. Adult rats were given either risperidone or
Available online 6 January 2006
olanzapine in their drinking water for 21 days. Memory was assessed on the first and last day of treatment using an object discrimination test, and the rate of cell proliferation in the
Keywords:
subventricular zone (SVZ), dentate gyrus (DG) and prefrontal cortex (PFC) was quantified by
Neuroleptic
immuno staining for Ki-67. The results show that both risperidone and olanzapine
Neurogenesis
significantly improved performance in object discrimination after 21 days, and
Cognition
additionally, olanzapine significantly increased cell proliferation in the SVZ and PFC but
Schizophrenia
not the DG. © 2005 Elsevier B.V. All rights reserved.
Adult brain
The continued proliferation of precursor cells which can generate new neurones in the adult brain has attracted increasing attention and is now thought to occur in the brains of all mammals, including man (for review, see Gould and Gross, 2002). Although many regions of the postnatal brain contain progenitor cells, capable of generating both neurones and glia, in vivo neurogenesis is restricted to a small number of specific regions. The largest of these is the subventricular zone (SVZ) of the lateral walls of the lateral ventricles (Brazel et al., 2003). Cells produced here have been shown, in rodents, to pass via the rostral migratory stream, to the olfactory bulbs where they differentiate into olfactory bulb granule and periglomerular neurones. Recent evidence now shows that the SVZ may also contribute new neurones to other brain regions (amygdala, cortex and striatum) in adult animals (Bernier et al., 2002; Dayer et al., 2005). The expression of the proteoglycan NG2 has been shown to be a marker for proliferating cells which can differentiate into neurones, and NG2+ cells are found both in the SVZ and in the cortex, raising the possibility that new neurones may be generated in situ at
the sites in which they will integrate (Dayer et al., 2005; Belachew et al., 2003). However, most evidence indicates that the low levels of cell proliferation which occur within the cerebral cortex, under non-pathological conditions, generate cells with a glial phenotype (Levison et al., 1999; Magavi et al., 2000). In agreement with this, recent studies have shown that treatment of animals with electroconvulsive seizures increases the numbers of proliferating cells in the frontal cortex, 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 the subgranular zone (SGZ), adjacent to the dentate gyrus (Gould et al., 2000; Cameron and McKay, 2001). Cells generated in the SGZ migrate into the dentate gyrus before differentiating into neurones and making functional connections. The study of cell proliferation in the adult brain has been stimulated by observations that the rate of cell division is affected by external influences and can be modulated by drug treatment. Interest has focused on the reduction in hippocampal neurogenesis, associated with depression and stress, and
⁎ Corresponding author. Fax: +44 115 9709 259. E-mail address: [email protected] (P.M. Wigmore).
0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.11.047
BR A I N R ES E A RC H 1 0 7 0 ( 2 00 6 ) 2 4 2 –2 45
its increase with chronic antidepressant treatment (Brown et al., 2003; Duman et al., 2001). The response to antidepressants requires the generation of new neurones (Santarelli et al., 2003), and it is possible that increases in cell proliferation are also associated with functional changes in other brain regions. Chronic administration of atypical neuroleptics has been reported recently to increase cell proliferation in the SVZ (Wakade et al., 2002) and cortex and hippocampus (Kodama et al., 2004). Schizophrenia is associated with a cognitive impairment and decreased neuronal function in the prefrontal cortex (PFC), (Friedman et al., 1999). Atypical neuroleptics are reported to improve cognition in schizophrenics, suggesting that 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 of atypical neuroleptics, we have examined the effect of chronic administration of olanzapine and risperidone on cognitive behavior and cell proliferation in the SVZ of the lateral ventricle, the SGZ of the hippocampus and the PFC. The object discrimination test was carried out on each of the control, risperidone and olanzapine treated groups (n = 6) at days 1 and 21 of the drug regimen. On day 1 (prior to drug treatment), there was no significant difference in discrimination scores between groups (data not shown). After 21 days of drug treatment, animals treated with either risperidone or olanzapine showed an increase in the time spent examining the novel object such that there was a significant increase in mean discrimination scores, compared to controls (Table 1). Ki-67 staining was carried out on 4 20-μm-thick brain sections per rat, for each of the prefrontal cortex, dentate gyrus and subventricular zones. All sections were 200 μm apart. This resulted in a sample size of 24 sections per treatment group for each area of the brain examined. Ki-67 positive cells were easily 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-67 positive cells. In the prefrontal cortex, there was no significant change in the number of Ki-67 positive cells following risperidone
Table 1 – Results of the object discrimination test and mean numbers of Ki-67 positive cells per section Discrimination Mean no. Mean Mean no. of Ki-67 ration of Ki-67 no. of positive positive Ki-67 cells per positive cells per section cells per section of SVZ of PFC section of dentate gyrus Control Risperidone treated Olanzapine treated
0.491 (0.085) 0.714 (0.052)* 0.778 (0.026)*
8.5 (0.99) 9.8 (1.08)
8.0 (1.03) 9.0 (2.18) 10.0 (0.97) 10.3 (0.99)
13.0 (1.53)* 10.8 (0.98) 16.2 (0.96)*
Figures in brackets indicate standard error of the mean, asterisk indicates significant difference from the controls.
243
Fig. 1 – Section through the dentate gyrus (star) stained with propidium iodide (red) to show all nuclei and Ki-67 (green). Ki-67 positive cells are seen in the subgranular zone adjacent to the dentate gyrus.
treatment, compared to controls. In contrast, there was a significant increase (44%) in the number of Ki-67 positive cells in the PFC of olanzapine treated rats, compared to controls (Table 1). Similarly in the subventricular zone, risperidone had no significant effect on the number of Ki-67 positive cells but olanzapine produced a significant increase in the number of positive cells in this region (Table 1). Finally in the dentate gyrus of the hippocampus, although a small increase in Ki-67 positive cells was seen with drug treatment, neither risperidone nor olanzapine had a significant effect on the number of Ki-67 positive cells (Table 1). The results show that both olanzapine and risperidone significantly improve object discrimination. Olanzapine significantly increased cell proliferation in the prefrontal cortex and subventricular zone. Cognitive impairment, which is considered to underlie schizophrenia, is linked to abnormalities within the PFC, and severe deficits in working memory are commonly observed in schizophrenic patients (Friedman et al., 1999; Nieoullon, 2002). For this reason, a measure of working memory is useful in assessing the action of neuroleptics. In the present study, treatment with two atypical antipsychotics, risperidone and olanzapine significantly improved working memory as indicated by the increased discrimination ratios in the object discrimination test. Olanzapine has been previously reported to increase cell proliferation 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 cell proliferation in this region after olanzapine treatment but not with risperidone. Under normal conditions, cells generated in the rodent SVZ, migrate rostrally and differentiate as neurones or supporting cells within the olfactory bulbs (Lois and Alvarez-Buylla, 1994; Lois et al., 1996; Luskin, 1993). In man, a similar proliferation of stem cells occurs in the lateral ventricle SVZ, and newly generated neurones are found in the olfactory bulbs (Sanai et al., 2004; Bedard and Parent,
244
BR A I N R ES E A RC H 1 0 7 0 ( 2 00 6 ) 2 4 2 –24 5
2004). Patients suffering from schizophrenia have reduced olfactory bulb and medial temporal lobe volumes which are in turn associated with impaired olfaction (Purdon and FlorHenry, 2000; Turetsky et al., 2000; Moberg and Turetsky, 2003; Turetsky et al., 2003). These anatomical and sensory deficits have been correlated with an increased proportion of immature neurones in the olfactory bulbs of schizophrenic patients and has led to the suggestion that a failure in the processes of neurodevelopment underlies aspects of this disease (Arnold et al., 2001). It is therefore of interest to note that olanzapine inhibits apoptosis and increases the proliferation of neural cells in vitro and stimulates nerve growth factor synthesis in vivo (Li et al., 1999; Dwyer et al., 2003; Parikh et al., 2004). It is however unclear what impact increased proliferation within the SVZ would have on either olfaction or the function of regions of cortex to which olfactory neurones project. A small but non-significant increase in cell proliferation in the present study was seen in the dentate gyrus of the hippocampus after treatment with risperidone and olanzapine, a result similar to that reported previously by Wakade et al. (2002). Other authors however have found significant increases in proliferation in this region with similar or higher doses of olanzapine (Wang et al., 2004; Kodama et al., 2004), suggesting that proliferating cells in this region respond to both atypical neuroleptics and antidepressants. The present study also found a significant increase in the numbers of dividing cells within the PFC after olanzapine but not risperidone treatment. Under normal physiological conditions, a small number of dividing cells can be found in this region which continue to generate astrocytes and oligodendrocytes throughout life (Levison et al., 1999; Magavi et al., 2000). Antidepressant drugs, electroconvulsive therapy and atypical antipsychotics all appear to act to increase proliferation in this region (Wang et al., 2004; Kodama et al., 2004; Madsen et al., 2005). When the differentiation of the newly generated cells has been followed, the results have shown that all new cells are non-neural (Kodama et al., 2004; Madsen et al., 2005). Changes in the PFC are of particular interest as alterations in this region are thought to underlie the cognitive deficits seen in schizophrenia. Patients suffering from schizophrenia have a reduced thickness of prefrontal cortex due to a reduced number of glia rather than changes in neuronal density (Sanfilipo et al., 2000; Cotter et al., 2001; Hof et al., 2003). This has led to the suggestion that abnormalities in glial cell function in this region may underlie some aspects of this disease. The finding in the present study that atypical neuroleptics significantly increase numbers of dividing cells in the PFC is in line with a report that prolonged antipsychotic treatment of primates increases the density of glia in the PFC (Selemon et al., 1999). In summary, anti-psychotics in the present study improved cognitive function and in the case of olanzapine, this was associated with increased proliferation in the PFC and SVZ. This study seeks to correlate the effects of antipsychotic drug administration on cognitive behavior and cell proliferation in different brain regions. Control (vehicle only) olanzapine and risperidone treated groups were randomly selected
from the same group of animals, and behavioral testing and histology were carried out on all animals. Adult male hooded Lister rats were supplied and housed in the Biomedical Services Unit, Queen's Medical Centre, University of Nottingham. The treatment of animals was in accordance with the Home Office guidelines on the use of animals for scientific research. Animals were housed 3 to a cage with a 12-h light/dark cycle and randomly assigned to Control (n = 6), risperidone (kindly donated by Janssen Pharmaceuticals, n = 6) and olanzapine (Eli Lilly Pharmaceuticals [n = 6]) treated groups. On day 1 of the experiment (prior to drug treatment), an object discrimination test was carried out on all animals as described below. All rats were allowed ad libitum access to food and provided with drug dissolved in drinking water. The fluid intake of each cage of rats was assessed by measuring the weight of the water bottles daily. Individual rats were estimated to drink ∼28 ml in a 24-h period, and the final concentration of each drug supplied in the drinking water was determined for each cage from the mean volumes consumed and mean bodyweight of the rats. Risperidone was administered at 0.5 mg/kg/day and olanzapine at 2 mg/ kg/day. These doses were chosen to correspond to recommended human doses (BNF 2002) and are identical to those used in previous studies (Gao et al., 1998; Wakade et al., 2002). Plasma concentrations of olanzapine administered at this dose by the same route have previously been measured and shown to be 9.3 ng/ml (Gao et al., 1998). The drug solutions were replaced every 3 days, and there was no difference in water intake between drug-administered and control groups. Rats were weighed at the start of the experiment and after each week, and there was no significant difference in weight between groups at any time. On day 21, the object discrimination test was repeated as described below. The method was modified from that described by Ennaceur and Meliani (1992) and Wasuntarawat (2001). The object discrimination test is an assessment of working memory, with no conditioning components and reference memory aspects (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 small plastic bottles, water-filled to ensure stability and decorated with colored tape to create individual visually distinct designs. Between tests, the equipment was cleaned with 10% ethanol to eliminate any odor cues. Rats were habituated to the testing box on the day prior to testing. After being exposed to identical bottles for 3 min, the animals experienced a 1-min retention delay before exposure to a both a familiar and novel object for 3 min. Object exploration was defined as directing the nose to the object at less than 2 cm. The discrimination ratio (DR) describes the difference in time spent exploring a novel and familiar object, divided by the total time spent exploring objects. In order to check whether drug administration had a general effect on exploratory behavior or locomotion, the total time spent exploring both novel and familiar objects was compared between control and drug treated animals. No significant difference was found between groups using a oneway ANOVA.
BR A I N R ES E A RC H 1 0 7 0 ( 2 00 6 ) 2 4 2 –2 45
Following the object discrimination test on day 21, all the rats were killed by rapid stunning followed by decapitation. The brains were removed and individual hemispheres placed in 30% sucrose on ice for 30 min before freezing in isopentane in liquid nitrogen. 20-μm cryostat (Microm) sections were cut and mounted on Apes coated slides. 5 coronal brain sections were collected every 400 μm and Bromomethyl blue staining was used to identify the PFC immediately rostral to the hippocampus, the dentate gyrus and the subventricular zone. For each hemisphere, 4 consecutive sections (each 400 μm apart) were selected using a systematic random sampling procedure (Mayhew and Burton, 1988) to assess cell proliferation in each of the regions (PFC, SVZ and SGZ). Cell proliferation was assayed by immunodetection of the Ki-67 protein, a marker of all stages of the cell cycle (Kee et al., 2002; Scholzen and Gerdes, 2000). Slides were immunostained for Ki-67 using a monoclonal antibody (Novocastra, UK) following the manufactures protocol. Sections were counter stained with propidium iodide (Sigma) to show all cell nuclei. The number of Ki-67 positive cells in the specific areas of interest from each section was recorded by the same investigator. Data analysis used Wilcoxon signed-rank test or one-way analysis of variance (ANOVA) with post hoc Kruskal–Wallis. REFERENCES
Arnold, S.E., Han, L.Y., Moberg, P.J., Turetsky, B.I., Gur, R.E., Trojanowski, J.Q., Hahn, C.G., 2001. Arch. Gen. Psychiatry 58, 829–835. Bedard, A., Parent, A., 2004. Brain Res. Dev. Brain Res. 151, 159–168. Belachew, S., Chittajallu, R., Aguirre, A.A., Yuan, X., Kirby, M., Anderson, S., Gallo, V., 2003. J. Cell Biol. 161, 169–186. Bernier, P.J., Bedard, A., Vinet, J., Levesque, M., Parent, A., 2002. Proc. Natl. Acad. Sci. U. S. A. 99, 11464–11469. Brazel, C.Y., Romanko, M.J., Rothstein, R.P., Levison, S.W., 2003. Prog. Neurobiol. 69, 49–69. Brown, J., Cooper-Kuhn, C.M., Kempermann, G., Van Praag, H., Winkler, J., Gage, F.H., Kuhn, H.G., 2003. Eur. J. Neurosci. 17, 2042–2046. Cameron, H.A., McKay, R.D., 2001. J. Comp. Neurol. 435, 406–417. Cotter, D.R., Pariante, C.M., Everall, I.P., 2001. Brain Res. Bull. 55, 585–595. Dayer, A.G., Cleaver, K.M., Abouantoun, T., Cameron, H.A., 2005. J. Cell Biol. 168, 415–427. Duman, R.S., Nakagawa, S., Malberg, J., 2001. Neuropsychopharmacology 25, 836–844. Dwyer, D.S., Lu, X.H., Bradley, R.J., 2003. Brain Res. 971, 31–39. Ennaceur, A., Meliani, K., 1992. Behav. Brain Res. 51, 83–92. Friedman, J.I., Temporini, H., Davis, K.L., 1999. Biol. Psychiatry 45, 1–16.
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Short Communication
Treatment with olanzapine increases cell proliferation in the subventricular 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 LE I N FO
AB S T R A C T
Article history:
The present study examines the effect of chronic treatment with two atypical neuroleptics,
Accepted 2 November 2005
commonly used to treat schizophrenia. Adult rats were given either risperidone or
Available online 6 January 2006
olanzapine in their drinking water for 21 days. Memory was assessed on the first and last day of treatment using an object discrimination test, and the rate of cell proliferation in the
Keywords:
subventricular zone (SVZ), dentate gyrus (DG) and prefrontal cortex (PFC) was quantified by
Neuroleptic
immuno staining for Ki-67. The results show that both risperidone and olanzapine
Neurogenesis
significantly improved performance in object discrimination after 21 days, and
Cognition
additionally, olanzapine significantly increased cell proliferation in the SVZ and PFC but
Schizophrenia
not the DG. © 2005 Elsevier B.V. All rights reserved.
Adult brain
The continued proliferation of precursor cells which can generate new neurones in the adult brain has attracted increasing attention and is now thought to occur in the brains of all mammals, including man (for review, see Gould and Gross, 2002). Although many regions of the postnatal brain contain progenitor cells, capable of generating both neurones and glia, in vivo neurogenesis is restricted to a small number of specific regions. The largest of these is the subventricular zone (SVZ) of the lateral walls of the lateral ventricles (Brazel et al., 2003). Cells produced here have been shown, in rodents, to pass via the rostral migratory stream, to the olfactory bulbs where they differentiate into olfactory bulb granule and periglomerular neurones. Recent evidence now shows that the SVZ may also contribute new neurones to other brain regions (amygdala, cortex and striatum) in adult animals (Bernier et al., 2002; Dayer et al., 2005). The expression of the proteoglycan NG2 has been shown to be a marker for proliferating cells which can differentiate into neurones, and NG2+ cells are found both in the SVZ and in the cortex, raising the possibility that new neurones may be generated in situ at
the sites in which they will integrate (Dayer et al., 2005; Belachew et al., 2003). However, most evidence indicates that the low levels of cell proliferation which occur within the cerebral cortex, under non-pathological conditions, generate cells with a glial phenotype (Levison et al., 1999; Magavi et al., 2000). In agreement with this, recent studies have shown that treatment of animals with electroconvulsive seizures increases the numbers of proliferating cells in the frontal cortex, 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 the subgranular zone (SGZ), adjacent to the dentate gyrus (Gould et al., 2000; Cameron and McKay, 2001). Cells generated in the SGZ migrate into the dentate gyrus before differentiating into neurones and making functional connections. The study of cell proliferation in the adult brain has been stimulated by observations that the rate of cell division is affected by external influences and can be modulated by drug treatment. Interest has focused on the reduction in hippocampal neurogenesis, associated with depression and stress, and
⁎ Corresponding author. Fax: +44 115 9709 259. E-mail address: [email protected] (P.M. Wigmore).
0006-8993/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.11.047
BR A I N R ES E A RC H 1 0 7 0 ( 2 00 6 ) 2 4 2 –2 45
its increase with chronic antidepressant treatment (Brown et al., 2003; Duman et al., 2001). The response to antidepressants requires the generation of new neurones (Santarelli et al., 2003), and it is possible that increases in cell proliferation are also associated with functional changes in other brain regions. Chronic administration of atypical neuroleptics has been reported recently to increase cell proliferation in the SVZ (Wakade et al., 2002) and cortex and hippocampus (Kodama et al., 2004). Schizophrenia is associated with a cognitive impairment and decreased neuronal function in the prefrontal cortex (PFC), (Friedman et al., 1999). Atypical neuroleptics are reported to improve cognition in schizophrenics, suggesting that 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 of atypical neuroleptics, we have examined the effect of chronic administration of olanzapine and risperidone on cognitive behavior and cell proliferation in the SVZ of the lateral ventricle, the SGZ of the hippocampus and the PFC. The object discrimination test was carried out on each of the control, risperidone and olanzapine treated groups (n = 6) at days 1 and 21 of the drug regimen. On day 1 (prior to drug treatment), there was no significant difference in discrimination scores between groups (data not shown). After 21 days of drug treatment, animals treated with either risperidone or olanzapine showed an increase in the time spent examining the novel object such that there was a significant increase in mean discrimination scores, compared to controls (Table 1). Ki-67 staining was carried out on 4 20-μm-thick brain sections per rat, for each of the prefrontal cortex, dentate gyrus and subventricular zones. All sections were 200 μm apart. This resulted in a sample size of 24 sections per treatment group for each area of the brain examined. Ki-67 positive cells were easily 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-67 positive cells. In the prefrontal cortex, there was no significant change in the number of Ki-67 positive cells following risperidone
Table 1 – Results of the object discrimination test and mean numbers of Ki-67 positive cells per section Discrimination Mean no. Mean Mean no. of Ki-67 ration of Ki-67 no. of positive positive Ki-67 cells per positive cells per section cells per section of SVZ of PFC section of dentate gyrus Control Risperidone treated Olanzapine treated
0.491 (0.085) 0.714 (0.052)* 0.778 (0.026)*
8.5 (0.99) 9.8 (1.08)
8.0 (1.03) 9.0 (2.18) 10.0 (0.97) 10.3 (0.99)
13.0 (1.53)* 10.8 (0.98) 16.2 (0.96)*
Figures in brackets indicate standard error of the mean, asterisk indicates significant difference from the controls.
243
Fig. 1 – Section through the dentate gyrus (star) stained with propidium iodide (red) to show all nuclei and Ki-67 (green). Ki-67 positive cells are seen in the subgranular zone adjacent to the dentate gyrus.
treatment, compared to controls. In contrast, there was a significant increase (44%) in the number of Ki-67 positive cells in the PFC of olanzapine treated rats, compared to controls (Table 1). Similarly in the subventricular zone, risperidone had no significant effect on the number of Ki-67 positive cells but olanzapine produced a significant increase in the number of positive cells in this region (Table 1). Finally in the dentate gyrus of the hippocampus, although a small increase in Ki-67 positive cells was seen with drug treatment, neither risperidone nor olanzapine had a significant effect on the number of Ki-67 positive cells (Table 1). The results show that both olanzapine and risperidone significantly improve object discrimination. Olanzapine significantly increased cell proliferation in the prefrontal cortex and subventricular zone. Cognitive impairment, which is considered to underlie schizophrenia, is linked to abnormalities within the PFC, and severe deficits in working memory are commonly observed in schizophrenic patients (Friedman et al., 1999; Nieoullon, 2002). For this reason, a measure of working memory is useful in assessing the action of neuroleptics. In the present study, treatment with two atypical antipsychotics, risperidone and olanzapine significantly improved working memory as indicated by the increased discrimination ratios in the object discrimination test. Olanzapine has been previously reported to increase cell proliferation 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 cell proliferation in this region after olanzapine treatment but not with risperidone. Under normal conditions, cells generated in the rodent SVZ, migrate rostrally and differentiate as neurones or supporting cells within the olfactory bulbs (Lois and Alvarez-Buylla, 1994; Lois et al., 1996; Luskin, 1993). In man, a similar proliferation of stem cells occurs in the lateral ventricle SVZ, and newly generated neurones are found in the olfactory bulbs (Sanai et al., 2004; Bedard and Parent,
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2004). Patients suffering from schizophrenia have reduced olfactory bulb and medial temporal lobe volumes which are in turn associated with impaired olfaction (Purdon and FlorHenry, 2000; Turetsky et al., 2000; Moberg and Turetsky, 2003; Turetsky et al., 2003). These anatomical and sensory deficits have been correlated with an increased proportion of immature neurones in the olfactory bulbs of schizophrenic patients and has led to the suggestion that a failure in the processes of neurodevelopment underlies aspects of this disease (Arnold et al., 2001). It is therefore of interest to note that olanzapine inhibits apoptosis and increases the proliferation of neural cells in vitro and stimulates nerve growth factor synthesis in vivo (Li et al., 1999; Dwyer et al., 2003; Parikh et al., 2004). It is however unclear what impact increased proliferation within the SVZ would have on either olfaction or the function of regions of cortex to which olfactory neurones project. A small but non-significant increase in cell proliferation in the present study was seen in the dentate gyrus of the hippocampus after treatment with risperidone and olanzapine, a result similar to that reported previously by Wakade et al. (2002). Other authors however have found significant increases in proliferation in this region with similar or higher doses of olanzapine (Wang et al., 2004; Kodama et al., 2004), suggesting that proliferating cells in this region respond to both atypical neuroleptics and antidepressants. The present study also found a significant increase in the numbers of dividing cells within the PFC after olanzapine but not risperidone treatment. Under normal physiological conditions, a small number of dividing cells can be found in this region which continue to generate astrocytes and oligodendrocytes throughout life (Levison et al., 1999; Magavi et al., 2000). Antidepressant drugs, electroconvulsive therapy and atypical antipsychotics all appear to act to increase proliferation in this region (Wang et al., 2004; Kodama et al., 2004; Madsen et al., 2005). When the differentiation of the newly generated cells has been followed, the results have shown that all new cells are non-neural (Kodama et al., 2004; Madsen et al., 2005). Changes in the PFC are of particular interest as alterations in this region are thought to underlie the cognitive deficits seen in schizophrenia. Patients suffering from schizophrenia have a reduced thickness of prefrontal cortex due to a reduced number of glia rather than changes in neuronal density (Sanfilipo et al., 2000; Cotter et al., 2001; Hof et al., 2003). This has led to the suggestion that abnormalities in glial cell function in this region may underlie some aspects of this disease. The finding in the present study that atypical neuroleptics significantly increase numbers of dividing cells in the PFC is in line with a report that prolonged antipsychotic treatment of primates increases the density of glia in the PFC (Selemon et al., 1999). In summary, anti-psychotics in the present study improved cognitive function and in the case of olanzapine, this was associated with increased proliferation in the PFC and SVZ. This study seeks to correlate the effects of antipsychotic drug administration on cognitive behavior and cell proliferation in different brain regions. Control (vehicle only) olanzapine and risperidone treated groups were randomly selected
from the same group of animals, and behavioral testing and histology were carried out on all animals. Adult male hooded Lister rats were supplied and housed in the Biomedical Services Unit, Queen's Medical Centre, University of Nottingham. The treatment of animals was in accordance with the Home Office guidelines on the use of animals for scientific research. Animals were housed 3 to a cage with a 12-h light/dark cycle and randomly assigned to Control (n = 6), risperidone (kindly donated by Janssen Pharmaceuticals, n = 6) and olanzapine (Eli Lilly Pharmaceuticals [n = 6]) treated groups. On day 1 of the experiment (prior to drug treatment), an object discrimination test was carried out on all animals as described below. All rats were allowed ad libitum access to food and provided with drug dissolved in drinking water. The fluid intake of each cage of rats was assessed by measuring the weight of the water bottles daily. Individual rats were estimated to drink ∼28 ml in a 24-h period, and the final concentration of each drug supplied in the drinking water was determined for each cage from the mean volumes consumed and mean bodyweight of the rats. Risperidone was administered at 0.5 mg/kg/day and olanzapine at 2 mg/ kg/day. These doses were chosen to correspond to recommended human doses (BNF 2002) and are identical to those used in previous studies (Gao et al., 1998; Wakade et al., 2002). Plasma concentrations of olanzapine administered at this dose by the same route have previously been measured and shown to be 9.3 ng/ml (Gao et al., 1998). The drug solutions were replaced every 3 days, and there was no difference in water intake between drug-administered and control groups. Rats were weighed at the start of the experiment and after each week, and there was no significant difference in weight between groups at any time. On day 21, the object discrimination test was repeated as described below. The method was modified from that described by Ennaceur and Meliani (1992) and Wasuntarawat (2001). The object discrimination test is an assessment of working memory, with no conditioning components and reference memory aspects (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 small plastic bottles, water-filled to ensure stability and decorated with colored tape to create individual visually distinct designs. Between tests, the equipment was cleaned with 10% ethanol to eliminate any odor cues. Rats were habituated to the testing box on the day prior to testing. After being exposed to identical bottles for 3 min, the animals experienced a 1-min retention delay before exposure to a both a familiar and novel object for 3 min. Object exploration was defined as directing the nose to the object at less than 2 cm. The discrimination ratio (DR) describes the difference in time spent exploring a novel and familiar object, divided by the total time spent exploring objects. In order to check whether drug administration had a general effect on exploratory behavior or locomotion, the total time spent exploring both novel and familiar objects was compared between control and drug treated animals. No significant difference was found between groups using a oneway ANOVA.
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Following the object discrimination test on day 21, all the rats were killed by rapid stunning followed by decapitation. The brains were removed and individual hemispheres placed in 30% sucrose on ice for 30 min before freezing in isopentane in liquid nitrogen. 20-μm cryostat (Microm) sections were cut and mounted on Apes coated slides. 5 coronal brain sections were collected every 400 μm and Bromomethyl blue staining was used to identify the PFC immediately rostral to the hippocampus, the dentate gyrus and the subventricular zone. For each hemisphere, 4 consecutive sections (each 400 μm apart) were selected using a systematic random sampling procedure (Mayhew and Burton, 1988) to assess cell proliferation in each of the regions (PFC, SVZ and SGZ). Cell proliferation was assayed by immunodetection of the Ki-67 protein, a marker of all stages of the cell cycle (Kee et al., 2002; Scholzen and Gerdes, 2000). Slides were immunostained for Ki-67 using a monoclonal antibody (Novocastra, UK) following the manufactures protocol. Sections were counter stained with propidium iodide (Sigma) to show all cell nuclei. The number of Ki-67 positive cells in the specific areas of interest from each section was recorded by the same investigator. Data analysis used Wilcoxon signed-rank test or one-way analysis of variance (ANOVA) with post hoc Kruskal–Wallis. REFERENCES
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