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Arrested neuronal proliferation and impaired hippocampal function following fractionated brain irradiation in the adult rat
Neuroscience 119 (2003) 635– 642
ARRESTED NEURONAL PROLIFERATION AND IMPAIRED HIPPOCAMPAL FUNCTION FOLLOWING FRACTIONATED BRAIN IRRADIATION IN THE ADULT RAT T. M. MADSEN,a P. E. G. KRISTJANSEN,b ¨ RTWEINa* T. G. BOLWIGa AND G. WO
survival of newborn neurons (Gould et al., 1999; Lemaire et al., 2000). Recently, Shors and colleagues reduced hippocampal neurogenesis in rats to 20% of control levels with an antimitotic agent (Shors et al., 2001). This was associated with impairment of hippocampus-dependent trace conditioning, while leaving hippocampus-independent delay conditioning unaffected. Newborn dentate granule neurons derive from a population of precursor or stem cells, residing in the subgranular zone (Gage et al., 1998). The majority of cells born in this region develop into neurons and migrate into the granule cell layer where they integrate into preexisting neuronal networks, forming connections characteristic of this region (Markakis and Gage, 1999). The time required for a newly born neuron to extend neurites and to attain the characteristics of a granule cell is estimated to be around 10 –14 days (Hastings and Gould, 1999). In another study, van Praag and colleagues showed that adult-generated neurons, labeled with a green fluorescent protein-retrovirus, display electrophysiological characteristics similar to mature granule cells 28 days after birth, and that these neurons have functional synapses at this time (van Praag et al., 2002). Ionizing irradiation has long been known to stop cellular proliferation. At appropriate doses it kills predominantly dividing cells, sparing neighboring, non-dividing cells. In the brains of adult rats, a single 10-Gy (absorbed dose) dose causes apoptosis among proliferating cells in the dentate gyrus of the hippocampus, leaving other cells unscathed (Peissner et al., 1999). A single 5-Gy dose was reported to block adult neurogenesis (Parent et al., 1999). In the clinic, fractionated whole-brain irradiation is used prophylactically to avoid metastasis to the CNS by eradication of micrometastatic deposits in the brain parenchyma (Auperin et al., 1999). In this study, we examined the effect of fractionated whole-brain irradiation on adult neurogenesis. Our goal was to characterize the consequences of this treatment for hippocampal function. Thus, we compared the performance of irradiated and non-irradiated rats in two tests of recognition memory, which differ with respect to their dependence on hippocampal function (M’Harzi et al., 1991; Steckler et al., 1998). Additionally we examined the animals’ activity level in an open field and their ability to learn the position of a hidden platform in a water maze. It is well established that lesions within the hippocampal formation are associated with changes in both those behaviors (Douglas and Isaacson, 1964; Morris et al., 1982; Walsh et al., 1986).
a
Laboratory of Neuropsychiatry, Department of Psychiatry O-6102, H:S Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark b Institute of Molecular Pathology, University of Copenhagen, Copenhagen, Denmark
Abstract—The generation of new neurons in the adult mammalian brain has been documented in numerous recent reports. Studies undertaken so far indicate that adult hippocampal neurogenesis is related in a number of ways to hippocampal function. Here, we report that subjecting adult rats to fractionated brain irradiation blocked the formation of new neurons in the dentate gyrus of the hippocampus. At different time points after the termination of the irradiation procedure, the animals were tested in two tests of short-term memory that differ with respect to their dependence on hippocampal function. Eight and 21 days after irradiation, the animals with blocked neurogenesis performed poorer than controls in a hippocampusdependent place-recognition task, indicating that the presence of newly generated neurons may be necessary for the normal function of this brain area. The animals were never impaired in a hippocampus-independent object-recognition task. These results are in line with other reports documenting the functional significance of newly generated neurons in this region. As our irradiation procedure models prophylactic cranial irradiation used in the treatment of different cancers, we suggest that blocked neurogenesis contributes to the reported deleterious side effects of this treatment, consisting of memory impairment, dysphoria and lethargy. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: adult neurogenesis, place recognition, object recognition, working memory, dentate gyrus, granule cell.
Adult hippocampal neurogenesis has been documented for a number of years in rodents, and has recently been demonstrated in several other mammalian species, including humans (Altman and Das, 1965; Eriksson et al., 1998; Kempermann et al., 1997). A number of studies relate increased hippocampal neurogenesis to increased demand on hippocampal function. In certain birds, food-storing behavior during the fall is associated with increased neurogenesis (Barnea and Nottebohm, 1994). When rats are trained in tasks, which require an intact hippocampus, there is a greater rate of neurogenesis and increased *Corresponding author. Tel: ⫹45-3545-6118; fax: ⫹45-3539-3546. E-mail address: [email protected] (G. Wo¨rtwein). Abbreviations: BrdU, bromodeoxyuridine; KPBS, potassium phosphate-buffered saline; KPBS-T, potassium phosphate-buffered saline⫹0.25% Triton X-100; PBS, phosphate-buffered saline.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00199-4
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EXPERIMENTAL PROCEDURES Animals and treatment Adult (approximately 2 month of age) male Wistar rats (M&B breeding center, Ll. Skensved, Denmark), weighing from 260 to 290 g at the beginning of the study were used. They were given free access to ordinary laboratory chow and water. All experimental procedures used in this experiment were in accordance with Danish Animal Experimentation Inspectorate (“Dyreforsøgstilsynet”) regulations. All efforts were made to minimize the number of animals used and their suffering.
Fractionated brain irradiation Under general anesthesia (Brietal; Eli Lilly, IN, USA; 40 mg/kg i.p.) the animals (n⫽27) were placed under a Stabilipan (Siemens, Munich, Germany) therapeutic unit (4.58 Gy/min at 300 kV, equipped with a Thoraeus I filter) and subjected to a 3-Gy irradiation dose directed at the hippocampal area. Doses used are calculated in the center of a 10 mm sphere. Irradiation was given during two 4-day periods separated by a 3-day pause. Controls (n⫽21) were anesthetized and handled as the experimental animals, but did not receive irradiation. No overt side effects of the treatment could be observed, and in a test arena, the animals displayed no abnormalities in locomotor behavior. However four of the irradiated and four control animals died under the effect of anesthesia during the 8 days of treatment.
Bromodeoxyuridine administration To study neurogenesis, the irradiated and control animals were subdivided into three groups and given two daily i.p. injections of the thymidine analog bromodeoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) (50 mg/kg) for 4 days. Animals were injected either during the second block of irradiation or on days 11–14 or on days 32–35 after the end of this treatment. Thus, one group of animals received BrdU injections during the course of irradiation, while the other groups were injected after the termination of this procedure, while experiencing various kinds of behavioral testing. BrdU is incorporated into DNA during the S-phase of the cell cycle and has a biological half-life of about 2 h. All groups were allowed to survive for 6 weeks after their last BrdU injection at which point the animals were transcardially perfused.
Behavioral testing Object recognition. This test was performed on days 5 and 18 after irradiation, essentially as described by Ennaceur and Delacour (Ennaceur and Delacour, 1988). The test was conducted in an open, black plastic box (76⫻56⫻40 cm). The objects to be discriminated were made of plastic and were of no natural significance to the animals. One of the objects existed in triplicate. Animals were habituated to the box for two 30-min sessions on the 2 days preceding the day of testing. During these habituation sessions no objects were present in the box. Twenty-four hours after the last habituation session a 3-min exploration trial was administered followed 15 min later by a 3-min discrimination trial. During the intertrial interval the animals were left undisturbed in their home-cages. During the exploration trial two identical objects were fixed with the help of Velcro tape to the center of two square areas, measuring 15 by 15 cm in the two back corners of the box. During the discrimination trial two new objects were presented in the same locations. One object was an identical copy of the two objects used during the exploration trial, while the other object was new. During the two 3-min trials the amount of time the animal spent exploring each object was registered using EthoVision software (Noldus Information Technology, Wageningen, The Netherlands). The software was used to delineate two “regions of interest”: the two square areas in the two back corners where the
objects were located. The time the animals’ center of gravity was found within either of the two “regions of interest” was registered by the software and used as a measure of object exploration. Trials during which the objects became dislodged from their regions of interest were excluded from analysis. The time spent exploring the novel object was expressed as percentage of the time spent exploring both objects during the discrimination trial and used as a measure of object recognition memory. The time spent exploring both objects during the exploration and discrimination trials was recorded and used as a measure of basal explorative behavior. Place recognition. This test was performed on days 8, 21, and 42 after irradiation, essentially as described previously (Cavoy and Delacour, 1993). The T-maze was constructed of gray laminated plywood with 30-cm-high walls. The stem measured 40 by 15 cm. A guillotine door could separate a 20-cm-long start box. The arms measured 30 by 15 cm and could be blocked of by guillotine doors. Conspicuous posters and objects were mounted on the walls of the room and left untouched throughout the experiment period. On the 2 days preceding the day of testing, the animals were habituated to the procedure by being placed into the start box with the guillotine door closed. After 15 s the door was removed and the animals were allowed to explore the apparatus for 5 min. During habituation both arms were freely accessible and the maze was placed in an enclosure, which blocked the view of the room. Twenty-four hours after the last of two habituation sessions, place recognition was assessed: at the start of each trial the animal was placed in the start box and 15 s later allowed to explore the maze for 3 min. During the first (exploration) trial one of the arms was blocked. During the second (discrimination) trial the animal could enter both arms. Exploration and discrimination trials were separated by an intertrial interval of 90 s, which the animal spent in its home cage. During the discrimination trial the amount of time the animal spent in each arm was registered using EthoVision software (Noldus Information Technology). An arm entry was registered when the center of gravity of the animal had crossed a line 15 cm from the center of the maze. The time spent in the novel arm was expressed as percentage of the time spent in both arms and used as a measure of place-recognition memory. Animals that did not leave the stem of the maze were excluded from statistical analysis. Locomotor activity. During the first habituation session for the object recognition tests (days 3 and 16 after irradiation) the activity of the animals was registered using EthoVision software (Noldus Information Technology). Activity level (total distance moved) was monitored for 30 min and analyzed for the total length of the session as well as in six 5-min intervals. Water-maze place learning. During the second week after the end of the irradiation procedure (days 10 –14) the ability to learn to navigate to a hidden platform in a water maze was examined in all the animals. This test was conducted essentially as described in Wo¨rtwein et al. (Wortwein et al., 1994). Briefly, the water maze (a circular pool measuring 200 cm in diameter with 50 cm high walls made of black plastic) was filled to 35 cm with room-temperature water and contained a submerged translucent platform. The platform remained in a fixed position in the center of one quadrant, 30 cm from the edge of the pool, throughout training and could not be seen by the swimming animal. Conspicuous posters and objects were mounted on the walls of the room. Rats were trained for 5 days, five trials per day. A trial consisted of placing the rat into the water maze at one of four randomly chosen start positions and allowing it to swim to and climb onto the platform, where it was left undisturbed for 30 s. After this, the animal was transferred to a transport cage, which was placed on a table a short distance from the pool while the experimenter prepared the next trial in the video tracking program (EthoVision,
T. M. Madsen et al. / Neuroscience 119 (2003) 635– 642 Noldus Information Technology). Then the animal was carried in the transport cage to the next start position and the next trial started. The experimenter guided animals to the platform, if they had not found it within 60 s.
Histology In order to obtain comparable brain sections for subsequent analysis, all animals were killed 6 weeks after their last BrdU injection. Thus, the animals were killed at different times after the behavioral tests. Rats were deeply anesthetized with Equithesin (SAD, Denmark, 3.3 mg/kg i.p.) and transcardially perfused with phosphatebuffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were left in the fixative overnight. Prior to sectioning into 40 m frontal sections on a freezing microtome, the brains were equilibrated in 30% sucrose in PBS. Every eighth section throughout the dorsal hippocampus starting with a randomly chosen section within the first eight was collected in cryoprotectant. BrdU-immunoperoxidase staining. Brains from all animals of all experimental groups were processed in parallel. Sections were stained free-floating and initially washed (3⫻10 min) in potassium PBS (KPBS). After an incubation in 1 M HCl at 65 °C for 30 min, sections were again washed in KPBS followed by incubation in blocking buffer (KPBS⫹0.25% Triton X-100⫹3% normal horse serum [Sigma, Vallensbæk Strand, Denmark] for 1 h. The primary antibody solution consisted of the blocking buffer⫹4% mouse anti-BrdU IgG (Becton-Dickinson, San Jose, CA, USA, no. 347580-7580), and the incubation took place for 36 h at 4 °C. After washing in KPBS⫹0.25% Triton X-100, the secondary antibody was applied (0.5% biotin horse–anti-mouse IgG in blocking buffer (Vector Laboratories, Burlingame, CA, USA, Vector BA-2001)) for 4 h at room temperature. Sections were then washed and incubated in avidin– biotin–peroxidase complex (Vectastain Elite ABC, Vector Laboratories) for 1 h, and peroxidase activity was visualized with diaminobenzidine (0.5 mg/ml, DAB Tablets, Kem-EnTec, Copenhagen, Denmark) and hydrogen peroxide. After rinsing, all sections were mounted, dehydrated, cleared in xylenes, and coverslipped. All slides were coded before counting. The total number of newly generated cells in the dentate gyrus of the dorsal hippocampus of each rat was estimated using a modified version of the fractionator method (West, 1993). Here, all BrdU-positive cells in the granule cell layer and subgranular zone of every eighth section throughout the entire dorsal hippocampus of both hemispheres were counted on an Olympus IX70 microscope, excluding the cells in the uppermost focal plane to avoid counting cell caps and focusing through the whole section. One animal from the irradiated group was excluded from this analysis due to failed perfusion fixation that resulted in high background staining. BrdU/NeuN double immunostaining. This was performed as described by Madsen et al. (Madsen et al., 2000). Sections were processed free-floating and initially washed in KPBS⫹0.25% Triton X-100 (KPBS-T). After a wash in 1 M HCl at 65 °C for 30 mins, sections were incubated in blocking buffer (KPBS-T⫹5% normal donkey serum, Harlan Sera-laboratory, Belton, UK; and 5% normal horse serum, Sigma, Vallensbæk Strand, Denmark). The primary antibody solution consisted of the blocking buffer⫹1% rat anti-BrdU (Harlan Sera-laboratory, MAS 250p)⫹1% mouse anti-NeuN (Chemicon, Temecula, CA, USA, MAB 377), and the incubation took place for 36 h at 4 °C. After another wash in blocking buffer, the secondary antibodies were applied (0.5% CY-3 donkey anti-rat IgG, Jackson Immunoresearch, West Grove, PA, USA, Jackson 712-165-153; and 0.5% biotin horse–antimouse IgG, Vector Laboratories, Vector BA-2001) for 2 h at room temperature, in the dark. Then the sections were incubated with
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0.4% fluorescine avidin D (Vector Laboratories, Vector A-2001) for 2 h. Finally, the sections were rinsed in KPBS, dried and mounted. To confirm the neuronal phenotype of the BrdU-labeled granular cells, sections were subjected to confocal microscopy on a Zeiss LSM 510 microscope. Split panel and z axis analysis were used for phenotypic characterization. Cells were examined manually in multi-channel configuration with a ⫻40 oil immersion objective and only cells in which the nucleus was clearly associated with both markers were considered newly generated neurons. Two non-irradiated animals and two animals from each of the two groups of irradiated animals that had received BrdU injections after the termination of the irradiation treatment were randomly selected for closer analysis. In those six animals 50 BrdU-positive cells from the hippocampal granule cell layer and the subgranular zone were scored for the presence of NeuN. Additionally, adjacent sections were stained with Cresyl Violet and examined microscopically for gross neuronal loss in the hippocampal formation and perihippocampal cortical areas.
Statistics Parameters are presented as means⫾S.E.M. The two experimental groups were compared by independent-samples t-tests. The percentage of total exploration time spent in the new arm during the place-recognition test and the percentage of total object exploration time spent on the new object during the object-recognition test were compared with chance (50%) for both experimental groups using one-sample t-tests. The level of significance was set at P⬍0.05.
RESULTS BrdU immunoreactivity In the hippocampi of rats that received BrdU injections and fractionated irradiation on the same days, virtually no BrdU-positive cells were present and this number was significantly lower than that observed in sham-treated controls (t10⫽⫺5.029, P⫽0,001). Also in the groups of animals that received injections of BrdU on days 11–14 and days 32–35 after irradiation, the number of BrdU-labeled cells was significantly lower than in the respective control groups (t12⫽⫺3.473, P⫽0,005 and t11⫽⫺6.826, P⫽0,00003, respectively). However, some level of proliferative activity had resumed (Fig. 1). Using confocal microscopy, it was established that many BrdU-labeled cells in the dentate gyrus also expressed the neuronal marker protein, NeuN, indicating a neuronal phenotype. Sections from all time points were analyzed, and, when present, some BrdU-positive cells in the granular layer also expressed NeuN, even in the irradiated animals (Fig. 2). In agreement with the immunoperoxidase studies, sections from animals that had received BrdU during the course of irradiation did not contain BrdU-labeled cells. The proportion of BrdU-positive cells that also expressed the neuronal marker NeuN was 79.6% and 80% in the two non-irradiated animals, 21.6% and 22% in the two animals that had received BrdU during the second week after irradiation, and 6% and 16% in the two animals that received BrdU during the fifth week after irradiation.
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Fig. 2. Confocal images. Both animals received the same dose of BrdU. Left panel: A section from the medial tip of the dentate gyrus of a control (non-irradiated) rat. Right panel: A section from an irradiated rat, which received BrdU during the irradiation procedure. NeuNlabeled cells (green) are seen in both sections, whereas BrdU-labeled cells (red) are seen only on the left panel. Scale bar in both⫽50 m.
In the object-recognition test both experimental groups discriminated equally well between a familiar and an unfa-
Fig. 1. Fractionated cranial irradiation reduces the number of BrdUpositive cells in the dorsal hippocampus dentate gyrus. Animals were treated with BrdU at different time points after irradiation. (A) received BrdU during the second block of irradiation (value for the irradiated animals: 2.22⫾2.22), (B) 2 weeks after the end of irradiation, (C) 6 weeks after the end of irradiation. Bars represent mean⫾S.E.M. and values for irradiated animals in A are too small to be seen.
Histology Microscopic examination of Nissl-stained adjacent sections did not reveal any gross neuronal loss in the hippocampal formation or perihippocampal cortical areas. Behavioral analyses There was no difference between irradiated and control groups in general locomotor activity level and habituation of activity over two 30-min test sessions administered on days 3 and 16 after cranial irradiation (Fig. 3).
Fig. 3. Locomotor activity. Animals were tested on two different occasions after brain irradiation. (A) During the first week after irradiation, and (B) 3 weeks after irradiation. No significant differences in the distance traveled during the 30-min test were observed. Bars represent mean⫾S.E.M.
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Fig. 4. Object recognition test. Animals were tested on two different occasions after brain irradiation. (A) During the first week after irradiation, and (B) 3 weeks after irradiation. No significant differences were observed. Bars represent mean⫾S.E.M. Both groups performed significantly above chance level (50%) during both tests.
miliar object at both test times (Fig. 4). The total time spent exploring objects did not differ between groups (data not shown). Both groups explored the new object significantly more than expected by chance at both test times (irradiated animals: t17⫽3.383, P⫽0.004, 5 days after irradiation and t22⫽5.851, P⫽0.000, 18 days after irradiation. Shamtreated animals: t13⫽4.580, P⫽0.001, 5 days after irradiation and t16⫽7.588, P⫽0.000, 18 days after irradiation). In the place-recognition test, the irradiated animals were impaired when tested in the first weeks after the end of irradiation treatment (Fig. 5). In the first and second place-recognition test, conducted on days 8 and 21 after termination of the irradiation procedure, the two experimental groups differed significantly with respect to the percentage of total arm exploration time spent in the new arm (t37⫽⫺2.167, P⫽0.037; t37⫽⫺2.245, P⫽0.024, respectively). In the third place-recognition test, conducted 42 days after irradiation, the two groups’ place recognition no longer differed significantly (t37⫽⫺0.748, P⫽0.459). The non-irradiated rats were always able to distinguish between the familiar and the newly opened arm. In all three
Fig. 5. Place-recognition test. (A) During the first week after irradiation a significant group difference in the preference for the newly opened arm of the T-maze is observed. (B) A significant group difference is still present 3 weeks after the end of irradiation. (C) No difference between the two groups 7 weeks after irradiation. Bars represent mean⫾S.E.M. During the second test irradiated animals’ performance did not differ significantly from chance (50%).
tests, conducted on days 8, 21, and 42 after irradiation, the control rats spent more than 50% of the total arm exploration time in the new arm (t16⫽6.035, P⫽0.000, t16⫽4.685, P⫽0.000, and t16⫽3.156, P⫽0.006, respectively). The irradiated rats displayed significant place recognition during the first and third, but not during the second test after irradiation (t21⫽3.572, P⫽0.002, t21⫽0.673, P⫽0.508, and t21⫽3.025, P⫽0.006, respectively).
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Fig. 6. Water-maze place-learning test. During the second week after the end of irradiation treatment the two groups did not differ in their ability to learn to navigate to a hidden platform. Shown is the mean latency to find the platform on five daily trials. Filled circles: control animals, open circles: irradiated animals. Error bars⫽S.E.M.
Also, in a standard place-learning test in a water maze, performed during the third week after the first block of irradiation, no significant group differences were found (Fig. 6).
DISCUSSION Here, we show that adult neurogenesis can be completely arrested by fractionated brain irradiation, but resumes somewhat within the following weeks. This halt in neurogenesis is associated with a deficit in a hippocampusdependent test of spatial short-term memory. The number of neurons generated daily in the dentate gyrus of the adult rat hippocampus has been disputed, but has been estimated to be up to 9000 under normal circumstances (Cameron and McKay, 2001) . However large, the number is still minute in comparison with the number of already existing granular neurons. In spite of this there are some indications that the continued supply of newborn neurons is important for certain aspects of normal hippocampal function. Recently Shors et al. (Shors et al., 2001) showed that animals with reduced dentate neurogenesis are impaired in a hippocampus-dependent traceconditioning task. Our data support and extend these findings: we show that rats with arrested cellular proliferation due to cranial irradiation do not perform as well as control animals in a task that draws on hippocampal function. Performance in the place-recognition test depends on proper processing of spatial cues, which, at least in the rodent, is dependent on intact hippocampal function (O’Keefe and Nadel, 1978). While place recognition is impaired after lesions in the hippocampal system (Jousselin-Hosaja et al., 1994; M’Harzi et al., 1991) such lesions
do not affect performance in the object-recognition test, whereas lesions in perihippocampal cortical areas do (Steckler et al., 1998). Our failure to find impaired performance in the object-recognition task therefore indicates that perihippocampal cortical areas function normally, despite the fact that the cortical mantle overlying the hippocampus had also been exposed to irradiation with the procedure used here. Lesions within the hippocampal formation are well known to cause hyperactivity and impair place learning in the water maze (Douglas and Isaacson, 1964; Morris et al., 1982; Walsh et al., 1986). Here we report that irradiation directed at the hippocampal area does neither cause hyperactivity nor have detrimental effects on the acquisition of a place-learning task in a water maze. This is consistent with our observation of preserved morphology on microscopic inspection. Rather, the selective and temporary suppression of neurogenesis seems to be associated with a category specific deficit in the temporary representation of an event, which is consistent with some theories of hippocampal function (Greenough et al., 1999). In other words, it appears that the presence of immature neurons in the dentate gyrus of the hippocampus is not necessary for normal locomotor activity levels or the learning of a spatial reference memory task, but seems to be required for a form of spatial memory that is much more transient. These results are in agreement with recently published data by Shors et al., who also failed to see differences in a water maze-based spatial learning test in rats with reduced neurogenesis. In that report, a change in trace fear conditioning was found to be associated with decreased neurogenesis (Shors et al., 2002). This apparent functional selectivity of newborn neurons in the adult dentate gyrus should be examined further. For example by comparing the performance of animals subjected to cranial irradiation in the water maze-based reference memory task to performance in a water maze-based working memory task, immediately after irradiation treatment, when practically no new neurons are present. Here, we did not conduct the water maze-based spatial reference memory task at the same time as the place-recognition task. However, place-recognition was impaired during both the week preceding and the week following water-maze training. In the adult rat, a single dose of 10 Gy induces apoptosis among the proliferating cells in the germinal zone of the subgranular zone (Peissner et al., 1999). In the subependyma, another area with a population of stem/precursor cells, apoptosis is observed after 2–3 Gy. Also, a reduction in cellular proliferation, measured by BrdU labeling, ensues 1–2 days after irradiation (Shinohara et al., 1997). Higher doses of irradiation result in larger numbers of apoptotic cells and a longer pause in proliferation (Shinohara et al., 1997). In a recent paper, Monje et al. (Monje et al., 2002), showed a marked decrease in newborn cells in the hippocampus 2 months after a 10-Gy dose. Not only is there a decrease in the number of both neurons and glia, but also a decrease in the relative proportion of neurons after irradiation. The authors ascribe this to a disruption of the microenvironment due to inflammatory processes and
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lack of proper microvasculature (Palmer et al., 2000). Here, we did not perform a comprehensive phenotypic analysis of the BrdU-labeled cells, although our observation of NeuN labeled BrdU-positive cells indicates that some of the newly generated cells in the subgranular zone and granular layer display a neuronal phenotype. This might in part be ascribed to us administering BrdU at different time points after irradiation. It is also possible that our fractionated dose regimen caused less damage to the neurogenic microenvironement than a single, relatively high dose. In any case, the number of BrdU-labeled cells in irradiated rats is only a fraction of the values seen in control animals. In the present study, we observe a complete suppression of BrdU incorporation when the thymidine analog is administered during the days when the animals were also irradiated. When BrdU is given at later stages, we observe a recurrence of some level of neurogenesis in the subgranular zone. This might indicate that with this fractionated dosage regimen of 3 Gy/day there is a population of surviving precursor cells with a capability to divide. There are now strong indications that the formation of new neurons in adulthood serves an important function. A substantial body of data couples an increase in neurogenesis with increased demand on the hippocampus (Barnea and Nottebohm, 1994; Gould et al., 1999; Lemaire et al., 2000). Furthermore, stress decreases hippocampal neurogenesis and impairs some forms of learning and memory while environmental enrichment increases neurogenesis and improves performance on certain learning and memory tasks (Kim and Diamond, 2002; van Praag et al., 2000). The present study and data from Shors et al. (Shors et al., 2001, 2002) indicate that some aspects of hippocampal performance are impaired when neurogenesis in this area is reduced. Also, the return of neurogenesis seems to be accompanied by recovery of function (Shors et al., 2001; present data). In a recent paper van Praag et al. show that newborn neurons in the dentate gyrus are capable of forming useful synapses that are electrophysiologically indistinguishable from older granule cells (van Praag et al., 2002). This would indicate that the new cells integrate into established networks, and consequently, attain functionality. The finding that new cells integrate into this network throughout life, and that this process is dependent on both genetic (Kempermann et al., 1997) and environmental (Gould et al., 1999; Kempermann et al., 2000; van Praag et al., 1999a,b) factors has led to hypotheses of a relationship between impairments in adult neurogenesis and the pathogenesis of depression and other stress-related disorders (Duman et al., 2000; Jacobs et al., 2000). The irradiation protocol used here is similar to a regimen used for prophylaxis of brain metastases from small-cell lung cancer (Kristjansen and Hansen, 1995). This treatment is associated with a number of side effects, some of which are confusion, depression, and memory disturbances. On the basis of our results, we propose that some of these side effects might be partially ascribed to disrupted neural proliferation.
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Acknowledgements—Birgit H. Hansen is thanked for expert technical assistance. Supported by The Ivan Nielsen foundation, the Theodore and Vada Stanley foundation, and a stipend from the NeuroScience PharmaBiotec Center (The Danish Medical Research Council) to G.W.
REFERENCES Altman J, Das GD (1965) Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. J Comp Neurol 124:319 –335. Auperin A, Arriagada R, Pignon JP, Le Pechoux C, Gregor A, Stephens RJ, Kristjansen PE, Johnson BE, Ueoka H, Wagner H, Aisner J (1999) Prophylactic cranial irradiation for patients with small-cell lung cancer in complete remission: Prophylactic Cranial Irradiation Overview Collaborative Group. N Engl J Med 341:476 – 484. Barnea A, Nottebohm F (1994) Seasonal recruitment of hippocampal neurons in adult free-ranging black-capped chickadees. Proc Natl Acad Sci USA 91:11217–11221. Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406 –417. Cavoy A, Delacour J (1993) Spatial but not object recognition is impaired by aging in rats. Physiol Behav 53:527–530. Douglas RJ, Isaacson RL (1964) Hippocampal lesions and activity. Psychon Sci 1:187–188. Duman RS, Malberg J, Nakagawa S, D’Sa C (2000) Neuronal plasticity and survival in mood disorders. Biol Psychiatry 48:732–739. Ennaceur A, Delacour J (1988) A new one-trial test for neurobiological studies of memory in rats: I. Behavioral data. Behav Brain Res 31:47–59. Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH (1998) Neurogenesis in the adult human hippocampus. Nat Med 4:1313–1317. Gage FH, Kempermann G, Palmer TD, Peterson DA, Ray J (1998) Multipotent progenitor cells in the adult dentate gyrus. J Neurobiol 36:249 –266. Gould E, Beylin A, Tanapat P, Reeves A, Shors TJ (1999) Learning enhances adult neurogenesis in the hippocampal formation. Nat Neurosci 2:260 –265. Greenough WT, Cohen NJ, Juraska JM (1999) New neurons in old brains: learning to survive? Nat Neurosci 2:203–205. Hastings NB, Gould E (1999) Rapid extension of axons into the CA3 region by adult-generated granule cells. J Comp Neurol 413:146 – 154. Jacobs BL, Praag H, Gage FH (2000) Adult brain neurogenesis and psychiatry: a novel theory of depression. Mol Psychiatry 5:262– 269. Jousselin-Hosaja M, Collery M, Delacour J (1994) Effects of adrenal medulla grafts on memory capacities of rats after hippocampal lesions. Neuroscience 59:275–284. Kempermann G, Kuhn HG, Gage FH (1997) Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proc Natl Acad Sci USA 94:10409 –10414. Kempermann G, van Praag H, Gage FH (2000) Activity-dependent regulation of neuronal plasticity and self repair. Prog Brain Res 127:35–48. Kim JJ, Diamond DM (2002) The stressed hippocampus, synaptic plasticity and lost memories. Nat Rev Neurosci 3:453–462. Kristjansen PE, Hansen HH (1995) Prophylactic cranial irradiation in small cell lung cancer: an update. Lung Cancer 12 (suppl 3):S23– S40. Lemaire V, Koehl M, Le Moal M, Abrous DN (2000) Prenatal stress produces learning deficits associated with an inhibition of neurogenesis in the hippocampus. Proc Natl Acad Sci USA 97:11032– 11037. M’Harzi M, Jarrard LE, Willig F, Palacios A, Delacour J (1991) Selec-
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tive fimbria and thalamic lesions differentially impair forms of working memory in rats. Behav Neural Biol 56:221–239. Madsen TM, Treschow A, Bengzon J, Bolwig TG, Lindvall O, Tingstrom A (2000) Increased neurogenesis in a model of electroconvulsive therapy. Biol Psychiatry 47:1043–1049. Markakis EA, Gage FH (1999) Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles. J Comp Neurol 406:449 –460. Monje ML, Mizumatsu S, Fike JR, Palmer TD (2002) Irradiation induces neural precursor-cell dysfunction. Nat Med 8:955–962. Morris RG, Garrud P, Rawlins JN, O’Keefe J (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297:681–683. O’Keefe J, Nadel L (1978) The hippocampus as a cognitive map. Oxford, UK: Clarendon. Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche for adult hippocampal neurogenesis. J Comp Neurol 425:479 –494. Parent JM, Tada E, Fike JR, Lowenstein DH (1999) Inhibition of dentate granule cell neurogenesis with brain irradiation does not prevent seizure-induced mossy fiber synaptic reorganization in the rat. J Neurosci 19:4508 –4519. Peissner W, Kocher M, Treuer H, Gillardon F (1999) Ionizing radiationinduced apoptosis of proliferating stem cells in the dentate gyrus of the adult rat hippocampus. Brain Res Mol Brain Res 71:61–68. Shinohara C, Gobbel GT, Lamborn KR, Tada E, Fike JR (1997) Apoptosis in the subependyma of young adult rats after single and fractionated doses of X-rays. Cancer Res 57:2694 –2702. Shors TJ, Miesegaes G, Beylin A, Zhao M, Rydel T, Gould E (2001)
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(Accepted 14 February 2003)
ARRESTED NEURONAL PROLIFERATION AND IMPAIRED HIPPOCAMPAL FUNCTION FOLLOWING FRACTIONATED BRAIN IRRADIATION IN THE ADULT RAT T. M. MADSEN,a P. E. G. KRISTJANSEN,b ¨ RTWEINa* T. G. BOLWIGa AND G. WO
survival of newborn neurons (Gould et al., 1999; Lemaire et al., 2000). Recently, Shors and colleagues reduced hippocampal neurogenesis in rats to 20% of control levels with an antimitotic agent (Shors et al., 2001). This was associated with impairment of hippocampus-dependent trace conditioning, while leaving hippocampus-independent delay conditioning unaffected. Newborn dentate granule neurons derive from a population of precursor or stem cells, residing in the subgranular zone (Gage et al., 1998). The majority of cells born in this region develop into neurons and migrate into the granule cell layer where they integrate into preexisting neuronal networks, forming connections characteristic of this region (Markakis and Gage, 1999). The time required for a newly born neuron to extend neurites and to attain the characteristics of a granule cell is estimated to be around 10 –14 days (Hastings and Gould, 1999). In another study, van Praag and colleagues showed that adult-generated neurons, labeled with a green fluorescent protein-retrovirus, display electrophysiological characteristics similar to mature granule cells 28 days after birth, and that these neurons have functional synapses at this time (van Praag et al., 2002). Ionizing irradiation has long been known to stop cellular proliferation. At appropriate doses it kills predominantly dividing cells, sparing neighboring, non-dividing cells. In the brains of adult rats, a single 10-Gy (absorbed dose) dose causes apoptosis among proliferating cells in the dentate gyrus of the hippocampus, leaving other cells unscathed (Peissner et al., 1999). A single 5-Gy dose was reported to block adult neurogenesis (Parent et al., 1999). In the clinic, fractionated whole-brain irradiation is used prophylactically to avoid metastasis to the CNS by eradication of micrometastatic deposits in the brain parenchyma (Auperin et al., 1999). In this study, we examined the effect of fractionated whole-brain irradiation on adult neurogenesis. Our goal was to characterize the consequences of this treatment for hippocampal function. Thus, we compared the performance of irradiated and non-irradiated rats in two tests of recognition memory, which differ with respect to their dependence on hippocampal function (M’Harzi et al., 1991; Steckler et al., 1998). Additionally we examined the animals’ activity level in an open field and their ability to learn the position of a hidden platform in a water maze. It is well established that lesions within the hippocampal formation are associated with changes in both those behaviors (Douglas and Isaacson, 1964; Morris et al., 1982; Walsh et al., 1986).
a
Laboratory of Neuropsychiatry, Department of Psychiatry O-6102, H:S Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen, Denmark b Institute of Molecular Pathology, University of Copenhagen, Copenhagen, Denmark
Abstract—The generation of new neurons in the adult mammalian brain has been documented in numerous recent reports. Studies undertaken so far indicate that adult hippocampal neurogenesis is related in a number of ways to hippocampal function. Here, we report that subjecting adult rats to fractionated brain irradiation blocked the formation of new neurons in the dentate gyrus of the hippocampus. At different time points after the termination of the irradiation procedure, the animals were tested in two tests of short-term memory that differ with respect to their dependence on hippocampal function. Eight and 21 days after irradiation, the animals with blocked neurogenesis performed poorer than controls in a hippocampusdependent place-recognition task, indicating that the presence of newly generated neurons may be necessary for the normal function of this brain area. The animals were never impaired in a hippocampus-independent object-recognition task. These results are in line with other reports documenting the functional significance of newly generated neurons in this region. As our irradiation procedure models prophylactic cranial irradiation used in the treatment of different cancers, we suggest that blocked neurogenesis contributes to the reported deleterious side effects of this treatment, consisting of memory impairment, dysphoria and lethargy. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: adult neurogenesis, place recognition, object recognition, working memory, dentate gyrus, granule cell.
Adult hippocampal neurogenesis has been documented for a number of years in rodents, and has recently been demonstrated in several other mammalian species, including humans (Altman and Das, 1965; Eriksson et al., 1998; Kempermann et al., 1997). A number of studies relate increased hippocampal neurogenesis to increased demand on hippocampal function. In certain birds, food-storing behavior during the fall is associated with increased neurogenesis (Barnea and Nottebohm, 1994). When rats are trained in tasks, which require an intact hippocampus, there is a greater rate of neurogenesis and increased *Corresponding author. Tel: ⫹45-3545-6118; fax: ⫹45-3539-3546. E-mail address: [email protected] (G. Wo¨rtwein). Abbreviations: BrdU, bromodeoxyuridine; KPBS, potassium phosphate-buffered saline; KPBS-T, potassium phosphate-buffered saline⫹0.25% Triton X-100; PBS, phosphate-buffered saline.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00199-4
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EXPERIMENTAL PROCEDURES Animals and treatment Adult (approximately 2 month of age) male Wistar rats (M&B breeding center, Ll. Skensved, Denmark), weighing from 260 to 290 g at the beginning of the study were used. They were given free access to ordinary laboratory chow and water. All experimental procedures used in this experiment were in accordance with Danish Animal Experimentation Inspectorate (“Dyreforsøgstilsynet”) regulations. All efforts were made to minimize the number of animals used and their suffering.
Fractionated brain irradiation Under general anesthesia (Brietal; Eli Lilly, IN, USA; 40 mg/kg i.p.) the animals (n⫽27) were placed under a Stabilipan (Siemens, Munich, Germany) therapeutic unit (4.58 Gy/min at 300 kV, equipped with a Thoraeus I filter) and subjected to a 3-Gy irradiation dose directed at the hippocampal area. Doses used are calculated in the center of a 10 mm sphere. Irradiation was given during two 4-day periods separated by a 3-day pause. Controls (n⫽21) were anesthetized and handled as the experimental animals, but did not receive irradiation. No overt side effects of the treatment could be observed, and in a test arena, the animals displayed no abnormalities in locomotor behavior. However four of the irradiated and four control animals died under the effect of anesthesia during the 8 days of treatment.
Bromodeoxyuridine administration To study neurogenesis, the irradiated and control animals were subdivided into three groups and given two daily i.p. injections of the thymidine analog bromodeoxyuridine (BrdU) (Sigma, St. Louis, MO, USA) (50 mg/kg) for 4 days. Animals were injected either during the second block of irradiation or on days 11–14 or on days 32–35 after the end of this treatment. Thus, one group of animals received BrdU injections during the course of irradiation, while the other groups were injected after the termination of this procedure, while experiencing various kinds of behavioral testing. BrdU is incorporated into DNA during the S-phase of the cell cycle and has a biological half-life of about 2 h. All groups were allowed to survive for 6 weeks after their last BrdU injection at which point the animals were transcardially perfused.
Behavioral testing Object recognition. This test was performed on days 5 and 18 after irradiation, essentially as described by Ennaceur and Delacour (Ennaceur and Delacour, 1988). The test was conducted in an open, black plastic box (76⫻56⫻40 cm). The objects to be discriminated were made of plastic and were of no natural significance to the animals. One of the objects existed in triplicate. Animals were habituated to the box for two 30-min sessions on the 2 days preceding the day of testing. During these habituation sessions no objects were present in the box. Twenty-four hours after the last habituation session a 3-min exploration trial was administered followed 15 min later by a 3-min discrimination trial. During the intertrial interval the animals were left undisturbed in their home-cages. During the exploration trial two identical objects were fixed with the help of Velcro tape to the center of two square areas, measuring 15 by 15 cm in the two back corners of the box. During the discrimination trial two new objects were presented in the same locations. One object was an identical copy of the two objects used during the exploration trial, while the other object was new. During the two 3-min trials the amount of time the animal spent exploring each object was registered using EthoVision software (Noldus Information Technology, Wageningen, The Netherlands). The software was used to delineate two “regions of interest”: the two square areas in the two back corners where the
objects were located. The time the animals’ center of gravity was found within either of the two “regions of interest” was registered by the software and used as a measure of object exploration. Trials during which the objects became dislodged from their regions of interest were excluded from analysis. The time spent exploring the novel object was expressed as percentage of the time spent exploring both objects during the discrimination trial and used as a measure of object recognition memory. The time spent exploring both objects during the exploration and discrimination trials was recorded and used as a measure of basal explorative behavior. Place recognition. This test was performed on days 8, 21, and 42 after irradiation, essentially as described previously (Cavoy and Delacour, 1993). The T-maze was constructed of gray laminated plywood with 30-cm-high walls. The stem measured 40 by 15 cm. A guillotine door could separate a 20-cm-long start box. The arms measured 30 by 15 cm and could be blocked of by guillotine doors. Conspicuous posters and objects were mounted on the walls of the room and left untouched throughout the experiment period. On the 2 days preceding the day of testing, the animals were habituated to the procedure by being placed into the start box with the guillotine door closed. After 15 s the door was removed and the animals were allowed to explore the apparatus for 5 min. During habituation both arms were freely accessible and the maze was placed in an enclosure, which blocked the view of the room. Twenty-four hours after the last of two habituation sessions, place recognition was assessed: at the start of each trial the animal was placed in the start box and 15 s later allowed to explore the maze for 3 min. During the first (exploration) trial one of the arms was blocked. During the second (discrimination) trial the animal could enter both arms. Exploration and discrimination trials were separated by an intertrial interval of 90 s, which the animal spent in its home cage. During the discrimination trial the amount of time the animal spent in each arm was registered using EthoVision software (Noldus Information Technology). An arm entry was registered when the center of gravity of the animal had crossed a line 15 cm from the center of the maze. The time spent in the novel arm was expressed as percentage of the time spent in both arms and used as a measure of place-recognition memory. Animals that did not leave the stem of the maze were excluded from statistical analysis. Locomotor activity. During the first habituation session for the object recognition tests (days 3 and 16 after irradiation) the activity of the animals was registered using EthoVision software (Noldus Information Technology). Activity level (total distance moved) was monitored for 30 min and analyzed for the total length of the session as well as in six 5-min intervals. Water-maze place learning. During the second week after the end of the irradiation procedure (days 10 –14) the ability to learn to navigate to a hidden platform in a water maze was examined in all the animals. This test was conducted essentially as described in Wo¨rtwein et al. (Wortwein et al., 1994). Briefly, the water maze (a circular pool measuring 200 cm in diameter with 50 cm high walls made of black plastic) was filled to 35 cm with room-temperature water and contained a submerged translucent platform. The platform remained in a fixed position in the center of one quadrant, 30 cm from the edge of the pool, throughout training and could not be seen by the swimming animal. Conspicuous posters and objects were mounted on the walls of the room. Rats were trained for 5 days, five trials per day. A trial consisted of placing the rat into the water maze at one of four randomly chosen start positions and allowing it to swim to and climb onto the platform, where it was left undisturbed for 30 s. After this, the animal was transferred to a transport cage, which was placed on a table a short distance from the pool while the experimenter prepared the next trial in the video tracking program (EthoVision,
T. M. Madsen et al. / Neuroscience 119 (2003) 635– 642 Noldus Information Technology). Then the animal was carried in the transport cage to the next start position and the next trial started. The experimenter guided animals to the platform, if they had not found it within 60 s.
Histology In order to obtain comparable brain sections for subsequent analysis, all animals were killed 6 weeks after their last BrdU injection. Thus, the animals were killed at different times after the behavioral tests. Rats were deeply anesthetized with Equithesin (SAD, Denmark, 3.3 mg/kg i.p.) and transcardially perfused with phosphatebuffered saline (PBS) followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were left in the fixative overnight. Prior to sectioning into 40 m frontal sections on a freezing microtome, the brains were equilibrated in 30% sucrose in PBS. Every eighth section throughout the dorsal hippocampus starting with a randomly chosen section within the first eight was collected in cryoprotectant. BrdU-immunoperoxidase staining. Brains from all animals of all experimental groups were processed in parallel. Sections were stained free-floating and initially washed (3⫻10 min) in potassium PBS (KPBS). After an incubation in 1 M HCl at 65 °C for 30 min, sections were again washed in KPBS followed by incubation in blocking buffer (KPBS⫹0.25% Triton X-100⫹3% normal horse serum [Sigma, Vallensbæk Strand, Denmark] for 1 h. The primary antibody solution consisted of the blocking buffer⫹4% mouse anti-BrdU IgG (Becton-Dickinson, San Jose, CA, USA, no. 347580-7580), and the incubation took place for 36 h at 4 °C. After washing in KPBS⫹0.25% Triton X-100, the secondary antibody was applied (0.5% biotin horse–anti-mouse IgG in blocking buffer (Vector Laboratories, Burlingame, CA, USA, Vector BA-2001)) for 4 h at room temperature. Sections were then washed and incubated in avidin– biotin–peroxidase complex (Vectastain Elite ABC, Vector Laboratories) for 1 h, and peroxidase activity was visualized with diaminobenzidine (0.5 mg/ml, DAB Tablets, Kem-EnTec, Copenhagen, Denmark) and hydrogen peroxide. After rinsing, all sections were mounted, dehydrated, cleared in xylenes, and coverslipped. All slides were coded before counting. The total number of newly generated cells in the dentate gyrus of the dorsal hippocampus of each rat was estimated using a modified version of the fractionator method (West, 1993). Here, all BrdU-positive cells in the granule cell layer and subgranular zone of every eighth section throughout the entire dorsal hippocampus of both hemispheres were counted on an Olympus IX70 microscope, excluding the cells in the uppermost focal plane to avoid counting cell caps and focusing through the whole section. One animal from the irradiated group was excluded from this analysis due to failed perfusion fixation that resulted in high background staining. BrdU/NeuN double immunostaining. This was performed as described by Madsen et al. (Madsen et al., 2000). Sections were processed free-floating and initially washed in KPBS⫹0.25% Triton X-100 (KPBS-T). After a wash in 1 M HCl at 65 °C for 30 mins, sections were incubated in blocking buffer (KPBS-T⫹5% normal donkey serum, Harlan Sera-laboratory, Belton, UK; and 5% normal horse serum, Sigma, Vallensbæk Strand, Denmark). The primary antibody solution consisted of the blocking buffer⫹1% rat anti-BrdU (Harlan Sera-laboratory, MAS 250p)⫹1% mouse anti-NeuN (Chemicon, Temecula, CA, USA, MAB 377), and the incubation took place for 36 h at 4 °C. After another wash in blocking buffer, the secondary antibodies were applied (0.5% CY-3 donkey anti-rat IgG, Jackson Immunoresearch, West Grove, PA, USA, Jackson 712-165-153; and 0.5% biotin horse–antimouse IgG, Vector Laboratories, Vector BA-2001) for 2 h at room temperature, in the dark. Then the sections were incubated with
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0.4% fluorescine avidin D (Vector Laboratories, Vector A-2001) for 2 h. Finally, the sections were rinsed in KPBS, dried and mounted. To confirm the neuronal phenotype of the BrdU-labeled granular cells, sections were subjected to confocal microscopy on a Zeiss LSM 510 microscope. Split panel and z axis analysis were used for phenotypic characterization. Cells were examined manually in multi-channel configuration with a ⫻40 oil immersion objective and only cells in which the nucleus was clearly associated with both markers were considered newly generated neurons. Two non-irradiated animals and two animals from each of the two groups of irradiated animals that had received BrdU injections after the termination of the irradiation treatment were randomly selected for closer analysis. In those six animals 50 BrdU-positive cells from the hippocampal granule cell layer and the subgranular zone were scored for the presence of NeuN. Additionally, adjacent sections were stained with Cresyl Violet and examined microscopically for gross neuronal loss in the hippocampal formation and perihippocampal cortical areas.
Statistics Parameters are presented as means⫾S.E.M. The two experimental groups were compared by independent-samples t-tests. The percentage of total exploration time spent in the new arm during the place-recognition test and the percentage of total object exploration time spent on the new object during the object-recognition test were compared with chance (50%) for both experimental groups using one-sample t-tests. The level of significance was set at P⬍0.05.
RESULTS BrdU immunoreactivity In the hippocampi of rats that received BrdU injections and fractionated irradiation on the same days, virtually no BrdU-positive cells were present and this number was significantly lower than that observed in sham-treated controls (t10⫽⫺5.029, P⫽0,001). Also in the groups of animals that received injections of BrdU on days 11–14 and days 32–35 after irradiation, the number of BrdU-labeled cells was significantly lower than in the respective control groups (t12⫽⫺3.473, P⫽0,005 and t11⫽⫺6.826, P⫽0,00003, respectively). However, some level of proliferative activity had resumed (Fig. 1). Using confocal microscopy, it was established that many BrdU-labeled cells in the dentate gyrus also expressed the neuronal marker protein, NeuN, indicating a neuronal phenotype. Sections from all time points were analyzed, and, when present, some BrdU-positive cells in the granular layer also expressed NeuN, even in the irradiated animals (Fig. 2). In agreement with the immunoperoxidase studies, sections from animals that had received BrdU during the course of irradiation did not contain BrdU-labeled cells. The proportion of BrdU-positive cells that also expressed the neuronal marker NeuN was 79.6% and 80% in the two non-irradiated animals, 21.6% and 22% in the two animals that had received BrdU during the second week after irradiation, and 6% and 16% in the two animals that received BrdU during the fifth week after irradiation.
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Fig. 2. Confocal images. Both animals received the same dose of BrdU. Left panel: A section from the medial tip of the dentate gyrus of a control (non-irradiated) rat. Right panel: A section from an irradiated rat, which received BrdU during the irradiation procedure. NeuNlabeled cells (green) are seen in both sections, whereas BrdU-labeled cells (red) are seen only on the left panel. Scale bar in both⫽50 m.
In the object-recognition test both experimental groups discriminated equally well between a familiar and an unfa-
Fig. 1. Fractionated cranial irradiation reduces the number of BrdUpositive cells in the dorsal hippocampus dentate gyrus. Animals were treated with BrdU at different time points after irradiation. (A) received BrdU during the second block of irradiation (value for the irradiated animals: 2.22⫾2.22), (B) 2 weeks after the end of irradiation, (C) 6 weeks after the end of irradiation. Bars represent mean⫾S.E.M. and values for irradiated animals in A are too small to be seen.
Histology Microscopic examination of Nissl-stained adjacent sections did not reveal any gross neuronal loss in the hippocampal formation or perihippocampal cortical areas. Behavioral analyses There was no difference between irradiated and control groups in general locomotor activity level and habituation of activity over two 30-min test sessions administered on days 3 and 16 after cranial irradiation (Fig. 3).
Fig. 3. Locomotor activity. Animals were tested on two different occasions after brain irradiation. (A) During the first week after irradiation, and (B) 3 weeks after irradiation. No significant differences in the distance traveled during the 30-min test were observed. Bars represent mean⫾S.E.M.
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Fig. 4. Object recognition test. Animals were tested on two different occasions after brain irradiation. (A) During the first week after irradiation, and (B) 3 weeks after irradiation. No significant differences were observed. Bars represent mean⫾S.E.M. Both groups performed significantly above chance level (50%) during both tests.
miliar object at both test times (Fig. 4). The total time spent exploring objects did not differ between groups (data not shown). Both groups explored the new object significantly more than expected by chance at both test times (irradiated animals: t17⫽3.383, P⫽0.004, 5 days after irradiation and t22⫽5.851, P⫽0.000, 18 days after irradiation. Shamtreated animals: t13⫽4.580, P⫽0.001, 5 days after irradiation and t16⫽7.588, P⫽0.000, 18 days after irradiation). In the place-recognition test, the irradiated animals were impaired when tested in the first weeks after the end of irradiation treatment (Fig. 5). In the first and second place-recognition test, conducted on days 8 and 21 after termination of the irradiation procedure, the two experimental groups differed significantly with respect to the percentage of total arm exploration time spent in the new arm (t37⫽⫺2.167, P⫽0.037; t37⫽⫺2.245, P⫽0.024, respectively). In the third place-recognition test, conducted 42 days after irradiation, the two groups’ place recognition no longer differed significantly (t37⫽⫺0.748, P⫽0.459). The non-irradiated rats were always able to distinguish between the familiar and the newly opened arm. In all three
Fig. 5. Place-recognition test. (A) During the first week after irradiation a significant group difference in the preference for the newly opened arm of the T-maze is observed. (B) A significant group difference is still present 3 weeks after the end of irradiation. (C) No difference between the two groups 7 weeks after irradiation. Bars represent mean⫾S.E.M. During the second test irradiated animals’ performance did not differ significantly from chance (50%).
tests, conducted on days 8, 21, and 42 after irradiation, the control rats spent more than 50% of the total arm exploration time in the new arm (t16⫽6.035, P⫽0.000, t16⫽4.685, P⫽0.000, and t16⫽3.156, P⫽0.006, respectively). The irradiated rats displayed significant place recognition during the first and third, but not during the second test after irradiation (t21⫽3.572, P⫽0.002, t21⫽0.673, P⫽0.508, and t21⫽3.025, P⫽0.006, respectively).
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Fig. 6. Water-maze place-learning test. During the second week after the end of irradiation treatment the two groups did not differ in their ability to learn to navigate to a hidden platform. Shown is the mean latency to find the platform on five daily trials. Filled circles: control animals, open circles: irradiated animals. Error bars⫽S.E.M.
Also, in a standard place-learning test in a water maze, performed during the third week after the first block of irradiation, no significant group differences were found (Fig. 6).
DISCUSSION Here, we show that adult neurogenesis can be completely arrested by fractionated brain irradiation, but resumes somewhat within the following weeks. This halt in neurogenesis is associated with a deficit in a hippocampusdependent test of spatial short-term memory. The number of neurons generated daily in the dentate gyrus of the adult rat hippocampus has been disputed, but has been estimated to be up to 9000 under normal circumstances (Cameron and McKay, 2001) . However large, the number is still minute in comparison with the number of already existing granular neurons. In spite of this there are some indications that the continued supply of newborn neurons is important for certain aspects of normal hippocampal function. Recently Shors et al. (Shors et al., 2001) showed that animals with reduced dentate neurogenesis are impaired in a hippocampus-dependent traceconditioning task. Our data support and extend these findings: we show that rats with arrested cellular proliferation due to cranial irradiation do not perform as well as control animals in a task that draws on hippocampal function. Performance in the place-recognition test depends on proper processing of spatial cues, which, at least in the rodent, is dependent on intact hippocampal function (O’Keefe and Nadel, 1978). While place recognition is impaired after lesions in the hippocampal system (Jousselin-Hosaja et al., 1994; M’Harzi et al., 1991) such lesions
do not affect performance in the object-recognition test, whereas lesions in perihippocampal cortical areas do (Steckler et al., 1998). Our failure to find impaired performance in the object-recognition task therefore indicates that perihippocampal cortical areas function normally, despite the fact that the cortical mantle overlying the hippocampus had also been exposed to irradiation with the procedure used here. Lesions within the hippocampal formation are well known to cause hyperactivity and impair place learning in the water maze (Douglas and Isaacson, 1964; Morris et al., 1982; Walsh et al., 1986). Here we report that irradiation directed at the hippocampal area does neither cause hyperactivity nor have detrimental effects on the acquisition of a place-learning task in a water maze. This is consistent with our observation of preserved morphology on microscopic inspection. Rather, the selective and temporary suppression of neurogenesis seems to be associated with a category specific deficit in the temporary representation of an event, which is consistent with some theories of hippocampal function (Greenough et al., 1999). In other words, it appears that the presence of immature neurons in the dentate gyrus of the hippocampus is not necessary for normal locomotor activity levels or the learning of a spatial reference memory task, but seems to be required for a form of spatial memory that is much more transient. These results are in agreement with recently published data by Shors et al., who also failed to see differences in a water maze-based spatial learning test in rats with reduced neurogenesis. In that report, a change in trace fear conditioning was found to be associated with decreased neurogenesis (Shors et al., 2002). This apparent functional selectivity of newborn neurons in the adult dentate gyrus should be examined further. For example by comparing the performance of animals subjected to cranial irradiation in the water maze-based reference memory task to performance in a water maze-based working memory task, immediately after irradiation treatment, when practically no new neurons are present. Here, we did not conduct the water maze-based spatial reference memory task at the same time as the place-recognition task. However, place-recognition was impaired during both the week preceding and the week following water-maze training. In the adult rat, a single dose of 10 Gy induces apoptosis among the proliferating cells in the germinal zone of the subgranular zone (Peissner et al., 1999). In the subependyma, another area with a population of stem/precursor cells, apoptosis is observed after 2–3 Gy. Also, a reduction in cellular proliferation, measured by BrdU labeling, ensues 1–2 days after irradiation (Shinohara et al., 1997). Higher doses of irradiation result in larger numbers of apoptotic cells and a longer pause in proliferation (Shinohara et al., 1997). In a recent paper, Monje et al. (Monje et al., 2002), showed a marked decrease in newborn cells in the hippocampus 2 months after a 10-Gy dose. Not only is there a decrease in the number of both neurons and glia, but also a decrease in the relative proportion of neurons after irradiation. The authors ascribe this to a disruption of the microenvironment due to inflammatory processes and
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lack of proper microvasculature (Palmer et al., 2000). Here, we did not perform a comprehensive phenotypic analysis of the BrdU-labeled cells, although our observation of NeuN labeled BrdU-positive cells indicates that some of the newly generated cells in the subgranular zone and granular layer display a neuronal phenotype. This might in part be ascribed to us administering BrdU at different time points after irradiation. It is also possible that our fractionated dose regimen caused less damage to the neurogenic microenvironement than a single, relatively high dose. In any case, the number of BrdU-labeled cells in irradiated rats is only a fraction of the values seen in control animals. In the present study, we observe a complete suppression of BrdU incorporation when the thymidine analog is administered during the days when the animals were also irradiated. When BrdU is given at later stages, we observe a recurrence of some level of neurogenesis in the subgranular zone. This might indicate that with this fractionated dosage regimen of 3 Gy/day there is a population of surviving precursor cells with a capability to divide. There are now strong indications that the formation of new neurons in adulthood serves an important function. A substantial body of data couples an increase in neurogenesis with increased demand on the hippocampus (Barnea and Nottebohm, 1994; Gould et al., 1999; Lemaire et al., 2000). Furthermore, stress decreases hippocampal neurogenesis and impairs some forms of learning and memory while environmental enrichment increases neurogenesis and improves performance on certain learning and memory tasks (Kim and Diamond, 2002; van Praag et al., 2000). The present study and data from Shors et al. (Shors et al., 2001, 2002) indicate that some aspects of hippocampal performance are impaired when neurogenesis in this area is reduced. Also, the return of neurogenesis seems to be accompanied by recovery of function (Shors et al., 2001; present data). In a recent paper van Praag et al. show that newborn neurons in the dentate gyrus are capable of forming useful synapses that are electrophysiologically indistinguishable from older granule cells (van Praag et al., 2002). This would indicate that the new cells integrate into established networks, and consequently, attain functionality. The finding that new cells integrate into this network throughout life, and that this process is dependent on both genetic (Kempermann et al., 1997) and environmental (Gould et al., 1999; Kempermann et al., 2000; van Praag et al., 1999a,b) factors has led to hypotheses of a relationship between impairments in adult neurogenesis and the pathogenesis of depression and other stress-related disorders (Duman et al., 2000; Jacobs et al., 2000). The irradiation protocol used here is similar to a regimen used for prophylaxis of brain metastases from small-cell lung cancer (Kristjansen and Hansen, 1995). This treatment is associated with a number of side effects, some of which are confusion, depression, and memory disturbances. On the basis of our results, we propose that some of these side effects might be partially ascribed to disrupted neural proliferation.
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Acknowledgements—Birgit H. Hansen is thanked for expert technical assistance. Supported by The Ivan Nielsen foundation, the Theodore and Vada Stanley foundation, and a stipend from the NeuroScience PharmaBiotec Center (The Danish Medical Research Council) to G.W.
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(Accepted 14 February 2003)