Increased neurogenesis in a rat ketamine model of schizophrenia

Increased Neurogenesis in a Rat Ketamine Model of Schizophrenia Gerburg Keilhoff, Hans-Gert Bernstein, Axel Becker, Gisela Grecksch, and Gerald Wolf Background: Growing evidence implicates abnormal neurodevelopment in schizophrenia, which manifests itself, for example, in reduced volume and cellular disarray of the hippocampus. This prompted us to investigate if there are indications of an altered neurodevelopment in this brain region. While neuron birth is largely completed by the end of gestation, granule neurons of the dentate gyrus are generated throughout life, thus offering an opportunity to investigate neurogenesis postnatally. Methods: We investigated whether repeated application of subanesthetic doses of the noncompetitive N-methyl-D-aspartate receptor antagonist ketamine, which has been shown to mimic model aspects of schizophrenia in animals, affects the hippocampal neurogenesis detected by bromodeoxyuridine incorporation. Cells were identified by immunocytochemistry. Results: Subanesthetic doses of ketamine applied subchronically enhance neurogenesis in the hippocampal subgranular zone. Conclusions: In our animal model of schizophrenia, ketamine may evoke its stimulating effect on neurogenesis via a block of the N-methyl-D-aspartate receptor directly by reducing the c-Fos/c-Jun expression, resulting in a depression of the AP1 transcription factor complex and/or by a reduced nitric oxide production or an enhanced serotonergic activity. The newly formed neurons are not able to overcome the schizophrenia-related loss of parvalbumin expressing neurons and the behavioral abnormalities indicating that their functional integration is crucial. Key Words: BrdU, hippocampus, immunocytochemistry, ketamine, neurogenesis, schizophrenia

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linical and postmortem research on the etiology and pathogenesis of schizophrenia implicates an abnormal development of neurons and synaptogenesis in quite different regions in schizophrenia (Harrison 1999). By the time the clinical expression of schizophrenia is apparent, neurogenesis and development are mostly complete. Granule neurons of the hippocampal dentate gyrus, however, are generated throughout life, thus offering an opportunity to investigate cellular and molecular events of neurogenesis and development at postnatal stages, even in adulthood. The importance of examining the hippocampal system in schizophrenia is underscored by the fact that abnormalities of the hippocampus, mainly a subtle but significant volume difference in schizophrenia, are one of the most consistent findings in schizophrenia research to date (Bogerts 1997; Heckers and Konradi 2002). This phenomenon was also demonstrated by brain imaging studies of patients with major depression (Bremner et al 2000), for which a reduced neurogenesis in the hippocampus was shown (Gould et al 1997). This apparent parallel prompted us to look for hippocampal neurogenesis in the ketamine model that closely resembles schizophrenia and represents a possible animal model in schizophrenia research (Becker et al 2003). Ketamine, a phencyclidine hydrochloride derivative and a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is able to induce in humans positive and negative symptoms similar to those associated with schizophrenia, including illusions, thought disorder and delusions, blunted emotional responses, emotional detachment, and psychomotor retardation From the Institute of Medical Neurobiology (GK, GW), Department of Psychiatry (H-GB), and Institute of Pharmacology and Toxicology (AB, GG), University of Magdeburg, Magdeburg, Germany. Address reprint requests to Dr. Gerburg Keilhoff, Institute of Medical Neurobiology, University of Magdeburg, Leipziger Strasse 44, D-39120 Magdeburg, Germany; E-mail: [email protected]. Received February 9, 2004; revised April 20, 2004; accepted June 7, 2004.

0006-3223/04/$30.00 doi:10.1016/j.biopsych.2004.06.010

(Bird et al 2001; Mechri et al 2001). The psychomimetic effects of ketamine are transitional, reversible, and influenced by time, dose, and administration conditions. Due to these characteristics, ketamine has recently been introduced as an animal model of schizophrenia (Duncan et al 2001; Miyamoto et al 2001; Becker et al 2003). Animals treated with ketamine developed a behavioral state that closely resembles schizophrenia, as previously demonstrated by data on face validity (changed social behavior, disruption of latent inhibition, enhanced responsiveness to MK-801 administration) and some aspects of construct validity (increased dopamine D2 receptor binding in the hippocampus, decreased glutamate binding in the frontal cortex, changes in dopamine and serotonin transporter systems) (Becker et al 2003). Moreover, the ketamine-treated animals showed remarkable cellular changes (increase in hippocampal expression of the calcium sensor protein visinin-like protein-1 [VILIP-1], reduction of parvalbumin-immunoreactive hippocampal interneurons) (Bernstein et al 2003; Keilhoff et al 2004), which, in part, resembled those observed in postmortem brains of schizophrenia patients. The similarity between these ketamine effects and related patients’ data support the idea that repeated administration of subanesthetic doses of ketamine might be a valuable tool in experimental schizophrenia research. Ketamine has been reported to primarily block the NMDA receptor complex (Kegeles et al 2000). In addition, single and repeated ketamine administration caused effects on dopamine, serotonin, and ␥-aminobutyric acid (GABA) transmission in the hippocampus (Irifune et al 1997). As the neurogenesis in the adult hippocampus can be influenced, among others, by the NMDA (Nacher et al 2003) as well as by the serotonergic (Arlotta et al 2003) system, there are at least two pathways via which ketamine can regulate cell proliferation and dysregulation, which are widely discussed as important pathogenetic factors in schizophrenia (Harrison 1999; Juckel et al 2003). Although the neurodevelopmental (prenatal and/or early postnatal) theory of schizophrenia is commonly accepted, MacCabe et al (2002) have postulated that a continuum view with a neurodevelopmental end on the one hand and a nonneurodevelopmental end on the opposite reflects the etiology of schizophrenia better than single narrow theories. This prompted us to use adult animals for the present study. BIOL PSYCHIATRY 2004;56:317–322 © 2004 Society of Biological Psychiatry

318 BIOL PSYCHIATRY 2004;56:317–322 Methods and Materials All experiments were performed in strict accordance with principles of laboratory animal care and the German law of the Protection of Animals. The present investigations were carried out using male Sprague-Dawley rats (Shoe:SPRD, Tierzucht Schönwalde GmbH, Schönwalde, Germany) aged 8 weeks (250 – 280 g). The animals were housed in groups of five under controlled laboratory conditions at 20°C ⫾ 2°C and air humidity of 55% to 60%, with a cycle of 12 hours light and 12 hours darkness (lights on at 6:00 AM). The animals had free access to commercial rat pellets (Altromin 1324, Altromin GmbH, Lage, Germany) and tap water. Animals in the experimental group (n ⫽ 14) received 30 mg/kg intraperitoneally injected ketamine (ketamine hydrochloride, Astrapin, Germany) (terminal half-time is 5.4 to 5 hours according to Hijazi et al 2003) dissolved in physiologic saline on five consecutive days. The control group I animals (n ⫽ 14) received saline at respective times, and the control group II animals (n ⫽ 5) were untreated. To assess cell proliferation, 33 rats were examined. An immunofluorescence assay for detection of 5-bromo-2=-deoxyuridine (BrdU) incorporated into cellular DNA (Boehringer Mannheim, Germany) in combination with immunofluorescence staining for cell identification was performed. Therefore, rats received in parallel to ketamine on 5 consecutive days one daily intraperitoneal injection of 50 mg/kg BrdU dissolved in physiologic saline. Two and 3 weeks after the cessation of ketamine/ saline treatment, animals (n ⫽ 7 per treatment at the respective time point) were anesthetized with chloral hydrate and transcardially perfused with 200 mL .1 mol/L phosphate buffered saline (PBS) (pH 7.4) and then at a rate of 15 mL/min with 400 mL 4% .1 mol/L phosphate buffered paraformaldehyde (pH 7.4) (Merk, Darmstadt, Germany). Brains were quickly removed from the cranium, postfixed in the same fixative at 4°C overnight, cryoprotected in a solution of 30% sucrose (Merk) in .4% buffered paraformaldehyde (pH 7.4) for 2 days, and rapidly frozen at ⫺20°C using 2-methylbutan (Roth, Karlsruhe, Germany). Serial frontal sections (20 ␮m thick) were cut on a cryostat (Jung Frigocut 2800 E, Leica, Bensheim, Germany). Free-floating sections were washed and incubated in 2N hydrochloric acid (HCl) for 1 hour at 37°C for DNA denaturation, neutralized by .1 mol/L borate buffer (pH 8.5), and incubated with a rat monoclonal antibody to BrdU (Oxford Biotechnology Ltd., Biozol, Eching, Germany) (1:100 in PBS containing .3% Triton X-100, Merck) for 1 hour at 37°C. After several rinses in PBS, slices were incubated with one of the following antibodies (working dilution optimum tested in preceding experiments) in PBS with .3% Triton X-100 and 1% normal goat serum overnight at 4°C: 1) polyclonal rabbit anti-NG2 chondroitin sulfate proteoglycan antibody (1:500) (Chemicon, Temecula, California); 2) polyclonal rabbit anti-glial fibrillary acidic protein (GFAP) (1:50) (Progen, Heidelberg, Germany); 3) monoclonal mouse anti-nestin (1:100) (Chemicon); 4) monoclonal mouse anti-NeuN (1:100) (Chemicon); 5) monoclonal mouse anti-microtubule-associated protein 2 (MAP2) (1:2500) (Sternberger Monoclonals Inc., Lutherville, Massachusetts); 6) monoclonal mouse anti-SMI 31 (1:5000) (Panneuronal neurofilament marker, Sternberger Monoclonals Inc.); and 7) polyclonal rabbit anti-parvalbumin (Ab-1) antibody (1:4000) (Oncogene Research Products, San Diego, California). Following incubation with primary antibodies, slices were washed in PBS (3 ⫻ 5 minutes) and incubated overnight with a combination of secondary antibodies (1:500) (Molecular Probes, www.elsevier.com/locate/biopsych

G. Keilhoff et al Göttingen, Germany): goat antirat-immunoglobulin G (IgG) Alexa Fluor 488/goat antirabbit-IgG Alexa Fluor 546 or goat antirat-IgG Alexa Fluor 546/goat antimouse-IgG Alexa Fluor 488; they were mounted and examined on a fluorescence microscope (Axiophot, Zeiss, Jena, Germany) equipped with phase-contrast, rhodamine optics or fluorescence images were collected using a Zeiss laser scanning microscope with a 63⫻ oil lens, using dual excitation at 488 and 543 nm and emission at 515 to 565 nm bandpass and long pass filter at 570 nm, respectively. Control reactions (substitution of the primary antisera by phosphate buffered saline) yielded negative results (i.e., no specific immunostaining was seen in these sections). To estimate the amount of BrdU-positive cells in the hippocampus, we manually counted stained cell profiles at higher magnification using the optical dissector method as described elsewhere (Bernstein et al 1998). A counting grid was used to obtain a reference square. At least 10 sections per hippocampus were counted. Since the thickness of the sections was only 20 ␮m, no infrasectional counting box was defined. The data were statistically analyzed by the nonparametric two-tailed U test (Mann–Whitney).

Results There was a basal level of BrdU immunolabeling in the hippocampus of untreated animals (control group II) that was consistent with previous reports (Kempermann and Gage 2000). Cells incorporated with BrdU were typically observed in the subgranular zone, on the border of the granule cell layer and the hilus, as well as within the hilus (Figure 1A). This is an area where neurogenesis normally persists in adult animals (Kempermann and Gage 2000). There were no obvious differences in the distribution of BrdU-immunoreactive cells at different longitudinal levels of the dentate gyrus. Neurogenesis Two Weeks After Ketamine Treatment Treatment with the NMDA receptor antagonist ketamine in subanesthetic doses induced a significant increase (100%) in the number of BrdU-labeled nuclei in the subgranular zone (Figure 1C) compared with saline-treated (Figure 1B) and untreated (Figure 1A) animals, as measured 2 and 3 weeks after the last BrdU injection (Figure 2). Independent of treatment, most of BrdU-labeled cells (⬃ 80%) had the morphology of granule cells (i.e., small round soma) and were co-immunostained for NeuN, a marker for mature neurons (Figure 1D). The main part of the remaining cells (10% of all BrdU-positive cells) had the morphology of granule cell precursors (round or oval, medium-sized cell bodies) and were labeled by nestin (Figure 1E, 1F), an intermediate filament that is expressed in neuronal stem or progenitor cells and thus can to some degree serve as a progenitor cell marker. Approximately 5% had the irregular or triangular morphologic characteristics of astroglial cells co-immunolabeled for GFAP (Figure 1G, 1H), a common marker for astroglial cells, and ⬃ 5% co-expressed the NG2 proteoglycan (Figure 1I), a marker for oligodendroglial precursor cells and/or synantocytes (Berry et al 2002). Despite the increase in cell number, the location of BrdU-labeled cells did not differ between ketamine-treated and saline-treated animals after a survival time of 2 weeks. There was no difference between the left and the right hippocampus, but significantly more BrdU-labeled cells were found in the lateral than in the medial blade of the dentate gyrus (Figure 1A-C). The newly formed cells were usually organized in clusters of 3 to 5 cells (Figure 1J).

G. Keilhoff et al

BIOL PSYCHIATRY 2004;56:317–322 319 Figure 1. BrdU immunohistochemistry in the dentate gyrus of adult rats. BrdU labeling of nuclei (green dots) of newly formed cells indicates in (A) baseline mitotic activity of an untreated animal, in (B) the unchanged BrdU incorporation 2 weeks after NaCl application, and in (C) the increased mitotic activity 2 weeks after application of ketamine in subanesthetic doses. (D-I) Identification of BrdU-labeled cells by double-immunofluorescence staining 2 weeks after ketamine treatment. (D, E) Co-localization of BrdU (red) with NeuN (green), a neuronal marker, is shown by yellow nuclei. (F, G) In some cases a double-staining of BrdU (green) and nestin (red), a marker of neuronal stem or progenitor cells, can be demonstrated. (H, I) BrdU (green) rarely co-localizes to the astrocytic marker GFAP (red) or (J) NG2 (red), a marker for oligodendroglial precursors and/or synantocytes. (K) Clustering of BrdU-positive nuclei (black) in the subgranular proliferative zone at the border of the hilus and granule cell layer 2 weeks after ketamine treatment, Nissl co-staining. (L) BrdU immunostaining (red) 3 weeks after ketamine treatment reveals a progressive dispersion of labeled cells throughout the granule cell layer, whereas the intensity of BrdU immunopositivity is reduced. (M) Co-localization of BrdU (red) and MAP2 (green), a marker of maturing neurons, is shown 2 weeks after ketamine treatment. (N) Newly formed neurons migrate into deeper zones of the granule cell layer along GFAP fibers 3 weeks after ketamine treatment. (O) Co-localization of BrdU (green) and parvalbumin (red) could not be shown (here 2 weeks after ketamine treatment). Scale bar: 100 ␮m in A-D; 50 ␮m in F, H, J, M, N, O; 25 ␮m in E, G, I, K, L. BrdU, 5-bromo-2=-deoxy-uridine; NaCl, sodium chloride; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; lb, lateral blade; mb, medial blade.

Neurogenesis Three Weeks After Ketamine Treatment In the group with a survival time of 3 weeks, single BrdUlabeled cells could be found additionally within the granule cell layer, indicating a discrete shift from the subgranular layer toward the middle part of the granule cell layer. Some of these cells were only moderately stained (Figure 1K), suggesting that they were derived from labeled precursor cells that had undergone several divisions, resulting in a dilution of BrdU-labeled DNA, or may possibly have resulted from less BrdU incorporation when cells were in late S phase at the time the BrdU was administered. These migrating cells co-expressed the neuronal marker MAP2 (Figure 1L) but in no case SMI 31 (a panneuronal neurofilament marker), indicating a state of relatively immaturity. They migrated along GFAP expressing fibers (Figure 1M). In other hippocampal regions, e.g., CA1 to CA3, BrdU immunoreactive neurons have never been observed. In no case could a co-staining of BrdU and parvalbumin be demonstrated (Figure 1N).

Discussion The key finding is that subanesthetic doses of ketamine, subchronically applied, enhance neurogenesis in the hippocampal subgranular zone. Most of the newly formed cells, detected by BrdU incorporation, were identified as neurons due to NeuN immunopositivity. The induction of hippocampal neurogenesis by ketamine is in contrast to predictions, because a loss of hippocampal neurons accompanied by a reduced hippocampal volume is an often-stated feature of schizophrenia (Harrison

1999). Some of these studies reviewed, however, may suffer from technical constraints, and several other investigators were not able to demonstrate any significant differences in number or density of neurons (Heckers et al 2002). Despite these discrepancies, there are convincing investigations showing that, at least, the density of the parvalbumin-immunoreactive subpopulation of GABAergic interneurons in the CA1 to CA3 hippocampal subfields in schizophrenics is selectively and substantially reduced (Zhang and Reynolds 2002). This observation coincides with our own findings in ketamine-treated rats reported previously (Keilhoff et al 2004). As we found in the present study, BrdU-labeling only in the dentate gyrus but never in the CA1 to CA3 region and, moreover, in no case a co-staining of BrdU and parvalbumin, our experiments give no evidence of a replacement of lost parvalbumin expressing GABAergic neurons in the hippocampus proper. This does not exclude the possibility that also inhibitory neurons (e.g., of the calretinin or calbindin subtype) occur among the newly formed cells, as was demonstrated by Liu et al (2003). Furthermore, we also cannot exclude that much longer survival times, as used in our study, could reveal migration and differentiation of newly formed neurons into parvalbumin-expressing cells. Replacement of CA1 neurons was reported in the case of global cerebral ischemia (Schmidt and Reymann 2002; Kokaia and Lindvall 2003). Global cerebral ischemia is well known to cause neurodegeneration in CA1 pyramidal neurons (Kokaia and Linvall 2003), whereas in case of diminished parvalbumin immunoreactivity as given in schizophrenics and in related animal models, it is thinkable that a decreased gene www.elsevier.com/locate/biopsych

320 BIOL PSYCHIATRY 2004;56:317–322

Figure 2. Quantitative analysis of ongoing cell proliferation in the dentate gyrus, as measured in rats injected with BrdU and sacrificed 2 and 3 weeks later, comparison of mean numbers (⫾ SEM) of BrdU immunoreactive nuclei in untreated, NaCl-treated, and ketamine-treated rats. The number of BrdU labeled cells is significantly increased (p ⬍ .005) in ketamine-treated rats compared with untreated and the respective NaCl-treated animals (nonparametric two-tailed U test, Mann–Whitney). BrdU, 5-bromo-2=-deoxy-uridine; NaCl, sodium chloride.

expression may look as if the number of GABAergic neurons is reduced. The mechanisms by which ketamine as a noncompetitive NMDA receptor antagonist may induce neurogenesis remain to be elucidated. There is evidence that blockade of NMDA receptors increases the number of newly generated cells in the rat brain (Gould et al 1997; Nacher et al 2003), which is in good agreement with our results. On the other hand, there are some reports that are contradictory to our findings. Luk et al (2003) showed that in utero exposure to NMDA receptor antagonists causes a dramatic reduction in striatal neurogenesis. Kitamura et al (2003) reported that, in mice, exercise-induced cellular proliferation in the hippocampus, but not basal proliferation, is dependent on NMDA receptors, at least on the ⑀1 subunit. These findings contradicting our present results and those of reports mentioned above might be explained by differences in the animal models (species, age, brain region, doses) used. We preferred a pharmacological, subchronical approach that avoids the possible phenomenon of compensation, as seen very often in knockout animal models. Moreover, regional variation in neurotransmitter effects on proliferation in the germinal zones of the telencephalon was proposed as an important mechanism for generating neuronal phenotypic diversity in the forebrain (Luk et al 2003), which may, in part, explain differences found in striatum versus hippocampus. One possible mechanism by which ketamine may influence cell proliferation via blocking the NMDA receptor directly is the attenuation of c-Fos and c-Jun expression leading to a depression of the AP1 transcription factor complex (Gerlach et al 2002). It appears that depression of the NMDA receptor modulates the de novo synthesis of these target proteins, which are responsible for proliferation of neural progenitor cells located in the dentate granular layer at that very level of gene transcription (Kitayama et al 2003). A second link between a blockade of NMDA receptors and induction of neurogenesis is offered by the NMDA-nitric oxide (NO) pathway. NO has been shown to negatively regulate mammalian adult neurogenesis (Gibbs 2003; Packer et al 2003). www.elsevier.com/locate/biopsych

G. Keilhoff et al Accordingly, a reduction in NO synthase (NOS) activity was shown by these authors to stimulate the production of new neurons in the hippocampal dentate gyrus. NO synthesis is well known to be enhanced by NMDA receptor activation via calcium influx that may activate the calcium/calmodulin-dependent NOS. A block of the NMDA receptor, as given here by ketamine, leads to a reduced NO production and may, in turn, result in an induction of neurogenesis. The molecular bases for the antiproliferative action of NO are not fully understood. Evidence from experimental models suggests that both cyclic guanine monophosphate (cGMP)-independent and cGMP-dependent mechanisms may be involved. One of such cGMP-independent cytostatic mechanisms is the inhibition of ornithine decarboxylase, whereby the polyamine synthesis is diminished, as ornithine decarboxylase is the initial and rate-limiting step in putrescine synthesis (Bernstein and Müller 1999). Lowering of cellular polyamine levels causes upregulation of p21, a universal inhibitor of the cell cycle (Bauer et al 2001). Alternatively, ketamine may act via the NMDA-serotonin pathway. Recent studies have provided evidence for a close relationship between NMDA receptor function and serotonergic activity (Breese et al 2002). For example, knocking out NMDA receptor function in mice increases the activity of the serotonergic system (Miyamoto et al 2001). Serotonin is known to possess a stimulating potency for granule cell production (Gould 1999; Dremencov et al 2003; Jacobs 2002), whereas a depletion of serotonin reduces neurogenesis (Brezun and Daszuta 1999). The hippocampus, especially the dentate gyrus, is the site of an extremely dense concentration of serotonin receptors (Kia et al 1996). Increased serotonergic activity is discussed as an important pathogenetic factor in schizophrenia (Juckel et al 2003), based on, among others, postmortem studies reporting altered densities of serotonergic receptors in schizophrenics (Harrison 1999), as well as on genetic (Eastwood et al 2001) and neuroimaging studies (Ngan et al 2000). Although our ketamine-treated rats showed an enhanced neurogenesis, they are characterized by a schizophrenia-like social behavior and a disruption of latent inhibition (Becker et al 2003). Moreover, the ketamine-treated animals show an increase in hippocampal expression of the calcium sensor protein VILIP-1 and a reduction of parvalbumin-immunoreactive hippocampal interneurons (Bernstein et al 2003; Keilhoff et al 2004). These observations led us to suggest that the enhanced neurogenesis is unable to overcome the ketamine-induced behavioral and neurochemical abnormalities that may result in schizophrenia-like phenomena. In relation to our results, a novel theory to provide the biological and cellular basis of major depression is highly interesting, as a failure of adult hippocampal neurogenesis has been suggested as an important depression-related pathogenetic factor (Jacobs 2002; Kempermann 2002; Kempermann and Kronenberg 2003). The authors have proposed that a reduced ability of the hippocampus to cope with novelty and complexity, leading to inadequate information processing at the interface between systems involved in learning and affect regulation, is a possible link between adult neurogenesis and depression. The insufficient reaction of patients to the challenges of the outside world could overwhelm the system and result in a hippocampal “shut down” (Kempermann and Kronenberg 2003). It is conceivable that such a psychic impairment, a typical feature also of schizophrenics (Harrison 1999), can result not only from a reduced cell production but also from an enhanced cell production. According to Arvidsson et al (2002) and Ekdahl et al (2002),

G. Keilhoff et al there are findings showing that in the adult brain neuronal self-repair by endogenous precursor cells may be functionally sufficient. Possibly, proliferation and/or differentiation of newly formed cells are disturbed and, subsequently, their integration into the respective brain areas. At any rate, precursors must interact with elements of the mature central nervous system (CNS) tissue, which will meet the newcomers as intruders. A high degree of degeneration of newly formed cells demonstrated by these authors probably reflects the unfavorable environment for the newcomers, which lack appropriate trophic support and connections. A similar scenario, i.e., decreased volume in combination with enhanced cell proliferation, was demonstrated for the olfactory epithelium in patients with schizophrenia (Turetsky et al 2000; Arnold et al 2001). These results were supported by in vitro studies showing that cell cultures derived from the olfactory epithelium of schizophrenic patients had higher mitotic indices compared with control subjects (Perry 2002). It is uncertain, so far, how growth, development, and behavior of olfactory receptor neurons are related to those of hippocampal neurons. The olfactory epithelium is embryologically closely related to the limbic region (Dryer and Graziadei 1994). It is derived from the olfactory placode that also generates some cells which migrate to the forebrain where they have been proposed to have a morphogenetic and inducing effect (Tarozzo et al 1995). Summing up, we were able to demonstrate an enhanced neurogenesis in the hippocampus of ketamine-treated rats. We hypothesize that in our animal model of schizophrenia, ketamine realizes its stimulating effect on hippocampal neurogenesis by blocking the NMDA receptor directly via reduction of c-Fos and c-Jun expression, leading to a depression of the AP1 transcription factor complex, and/or via a reduced NO production or an enhanced serotonergic activity. Both phenomena are reported to enhance the hippocampal neurogenesis. The newly formed neurons, however, are not able to overcome the schizophreniarelated loss of parvalbumin-expressing neurons in the hippocampus, a brain region that is suggested to be necessary for coping with challenges of the outside world. That may be causally related to an incorrect integration of the newly formed cells into the well-established orchestra of adult brain cells rather than a general neuronal loss. This study was supported by the Bundesministerium für Bildung und Forschung of Germany (NBL3) and the HertieStiftung (1.01.1/03/011). The skilled technical assistance of Leona Bück and Regina Domm is gratefully acknowledged. Arlotta P, Magavi SS, Macklis JD (2003): Induction of adult neurogenesis: Molecular manipulation of neural precursors in situ. Ann N Y Acad Sci 991:229 –236. Arnold SE, Han LY, Moberg PJ, Turetsky BI, Gur RE, Trojanowski JQ, et al (2001): Dysregulation of olfactory receptor neuron lineage in schizophrenia. Arch Gen Psychiatry 58:829 – 835. Arvidsson A, Collin T, Kirik D, Kokaia Z, Linvall O (2002): Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 8:963–970. Bauer PM, Buga GM, Ignarro LJ (2001): Role of p42/p44 mitogen-activatedprotein kinase and p21waf1/cip1 in the regulation of vascular smooth muscle cell proliferation by nitric oxide. Proc Natl Acad Sci U S A 98: 12802–12807. Becker A, Peters B, Schroeder H, Mann T, Huether G, Grecksch G (2003): Ketamine-induced changes in rat behaviour: A possible animal model of schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 27:687–700. Bernstein H-G, Becker A, Keilhoff G, Spilker C, Gorczyca A, Braunewell K-H, et al (2003): Brain region-specific changes in the expression of calcium

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