Acta Physiologica Hungarica, Volume 99 (2), pp. 126–132 (2012) DOI: 10.1556/APhysiol.99.2012.2.5
Effects of prenatal irradiation on behaviour and hippocampal neurogenesis in adult rats L Tomášová, B Šmajda, J Ševc Institute of Biology and Ecology, Faculty of Science, P. J. Šafárik University in Košice, Košice, Slovak Republic Received: March 31, 2011 Accepted after revision: January 23, 2012 Prenatal irradiation is known to have aversive effects on the brain development, manifested in changes in some behavioural parameters in adult individuals. The aim of our work was to assess the effect of prenatal irradiation on different forms of behaviour and on hippocampal neurogenesis in rats. Pregnant female rats were irradiated with a dose of 1 Gy of gamma rays on the 16th day of gravidity. The progeny of irradiated and control animals aged 3 months were tested in Morris water maze (MWM), open field (OF) and in elevated plus maze test (PM). The prenatal irradiation negatively influenced the short-term spatial memory in MWM in female rats, although the long-term memory was not impaired. A statistically significant increase of basic locomotor activity in OF was observed in irradiated rats. The comfort behaviour was not altered. The results of PM showed an increase of anxiety in irradiated females. The level of hippocampal neurogenesis, assessed as the number of cells labelled with 5-bromo-2deoxyuridine in the area of gyrus dentatus, was not statistically different in irradiated rats. Our results indicate, that prenatal irradiation with a low dose of gamma-rays can affect some innate and learned forms of behaviour in adult rats. We did not confirm a relation of behavioural changes to the changes of hippocampal neurogenesis. Keywords: prenatal irradiation, behaviour, hippocampal neurogenesis, rat, pregnant rats, Morris water maze, locomotor activity, gyrus dentatus
The results of animal experiments, experiences from the use of ionizing radiation in health care (4) and from irradiation of people in nuclear accidents (18) as well, have shown that ionizing radiation applied during pregnancy has a very negative impact on the developing central nervous system. Prenatal irradiation can interfere with the development of brain in a variety of ways and may alter its normal development and function, which, in turn, can lead to behavioural disorders in the adult age (9). The hippocampus is critical in the integration of neural processes involved in emotional behaviour, learning and memory (13). It has been established recently, that in the area of gyrus dentatus of this brain structure new neurons are produced throughout the life (1, 5). In some studies a close connection between the dose of prenatal irradiation, the decrease of mitotic activity in the hippocampus and alterations of behaviour in adult animals were found (9, 17, 16), but these relations are still not fully understood (3). The main problem, we aimed to address in our experiments was, whether low doses of irradiation, applied during the embryonic development of the individual, would influence mental processes in the adult age and whether structural changes in the brain could be detected. Behavioural changes in the adult age (e.g. deprived learning ability, changes in motoric and emotional responses) after exposure to very low prenatal or perinatal doses represent a serious problem in human medicine (e.g. in radiotherapy of brain tumours in children).
Corresponding author: Prof. Beňadik Šmajda E-mail:
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Materials and Methods Animals and irradiation procedure Wistar strain rats were used in the experiments. The animals were housed in plastic cages, under controlled environmental conditions with artificial light regimen (12:12 h light/dark cycles with light on at 7:00). They were fed with a standard laboratory diet and tap water ad libitum. Four-month-old virgin females (n = 2) were allowed to mate with males of the same age. Pregnant females were exposed to a dose of 1 Gy of gamma-rays from a 60Co-source th (Chisostat apparatus, Chirana, Czech Republic) on the 16 day of gravidity. The progeny of irradiated mothers (14 males and 9 females) and of shame-irradiated mothers (9 males and 9 females) were housed under standard conditions. Three months-old rats of both sexes were behaviourally tested in Morris water maze (MWM), in open field (OF) and in plus maze test (PM). The behaviour of animals during testing was recorded and evaluated using the computerized video-tracking system Smart Junior (Panlab, Barcelona, Spain). Morris water maze Spatial memory and learning of animals was followed in this test. The apparatus consisted of a plastic circular pool (100 cm diameter and 50 cm high) filled with water of 32 °C up to 20 cm of height. An escape platform, sized 15 × 15 cm was placed 2 cm below the water surface. The water was made opaque by addition of powdered milk. A day before the start of the experiment, the animals were habituated to the maze (without platform) for 2 min. After the first trial the test was repeated after 24 hours (testing of the short-term memory) and later after 1 week (testing of the long-term memory). The time needed to find the hidden platform and the length of swimming-track were recorded. The open field test The open field test was used to measure the locomotor activity and the comfortable behaviour. An open field seized 100 × 100 cm was used, bounded by white wooden walls of 60 cm high. The test was carried out on 5 consecutive days. At the start of the test the rat was placed into the centre of the field. Each trial lasted 8 min. During this time the locomotor activity (given as the distance walked in cm) and the comfort behaviour (given as the number of “face washing” acts) were recorded. The plus maze test The level of anxiety and the exploratory behaviour were measured in this test. An apparatus with two open and two closed arms of the length of 80 cm and a central square sized 15 x 15 cm was used. The arms of the maze were in a height of 55 cm above the ground. Each animal was placed in the central square of the maze and during 5 min the time spent in the open arms of apparatus and the exploratory activity (number of rearing) were recorded. Assessment of neurogenesis After finishing the behavioural tests the level of hippocampal neurogenesis in irradiated and control rats was assessed. To label the proliferating cells in the hippocampus the animals received i.p. injections of 5-bromo-2-deoxyuridine (BrdU, Sigma) in a daily dose of 100 mg/1 kg daily for 3 consecutive days. Two hours after the last injection rats were anesthetized and perfused transcardially with 0.9% saline followed by 4% fresh paraformaldehyde solution. The brain was removed, postfixed for 24 hours in 4% paraformaldehyde and transferred to 30% sucrose solution for cryopreservation. Consecutively, 40 μm-thick coronal Acta Physiologica Hungarica 99, 2012
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sections were cut on cryostat. The sections were incubated in 2M HCl for 30 min at 59 °C and rinsed in 0.1 M boric buffer, pH 8.5 for 30 min. Endogenous peroxidase reactivity was inhibited by 20% methanol containing 0.3% hydrogen peroxide for 30 min. Nonspecific protein activity was blocked in PBS containing 10% normal goat serum (NGS, Vector Laboratories, Burlingame, CA) and 0.3% Triton-X 100, for 30 min at room temperature. Then the sections were incubated with monoclonal rat primary antibody against BrdU (1:400, AbD Serotec, UK) in 5% NGS and 0.3% Triton-X 100 in PBS at 4 °C overnight followed by 2 h in goat anti rat IgG biotinylated secondary antibody (1:200, Vector Laboratories, Burlingame, CA) in 5% NGS and 0.3% Triton-X 100 at room temperature. Sections were incubated with avidin-biotin peroxidase system (ABC, Vector Laboratories, Burlingame, CA) for 1 h at room temperature. Antibody binding was detected by incubation with 3´,3´-diaminobenzidine (DAB, Vector Laboratories, Burlingame, CA). Sections were mounted onto gelatin-coated glass slides, dehydrated and coverslipped using Vectamount (Vector Laboratories, Burlingame, CA). Finally, the total number of BrdU-positive cells per 1 mm3 of tissue in the area of gyrus dentatus was counted using the software QuickPHOTO Micro 2.2 and Ellipse 2. Statistics The experimental data were evaluated using two-way ANOVA and the Tukey´s HSD test for behavioural parameters and the unpaired t-test for the cell numbers in neurogenesis. Results Morris water maze The results of the Morris water maze test confirmed partially, that the applied dose of ionizing radiation could have negative effects on learning and spatial memory: at 2nd trial (24 hours after first trial) irradiated rats needed longer distance to swim then controls to reach the platform (P = 0.0253). This difference was more pronounced in females, than in males (P = 0.0268, Fig. 1). The results for the latency time (Fig. 2) showed the same tendency, but the differences were not statistically significant (P = 0.1136 and P = 0.3235, respectively). No statistically significant effect of radiation on the long-term memory (3rd trial) was detected in either of the sexes (Figs 1 and 2). The open field test The results of this test showed that locomotor activity, expressed as the distance crossed during the test was in general higher in irradiated animals of both sexes, reaching statistical significance on the 2nd (P = 0.052) and on the 3rd (P = 0.019) experimental days (Fig. 3). The locomotor acitivity of females was significantly higher, than of males during the whole experiment, except the 1st day. The comfort activity (washing) was not significantly changed (Fig. 4). The plus maze The irradiated animals spent less time in the open arms of the maze, then did the controls (P = 0.015) with significant difference in males (P < 0.05). Significant intersexual differences were detected in the number of rearings with higher score in females (P = 0.045). None of the other comparisons were statistically significant (Table I). Acta Physiologica Hungarica 99, 2012
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Fig. 1. Learning in Morris water maze in prenatally irradiated and control rats, given as the swimming distance ± SEM to reach the platform; ** P < 0.01
Fig. 2. Learning in Morris water maze in prenatally irradiated and control rats, given as the latency time ± SEM to reach the platform; * P < 0.05
Fig. 3. Locomotor activity in open field test in prenatally irradiated and control rats, expressed as the total distance crossed during the test ± SEM. * P < 0.05; ** P < 0.01
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Fig. 4. “Face washing” behaviour in open field test in prenatally irradiated and control rats, expressed as the average number of cleaning acts ± SEM
Table I. Selected behavioural parameters of prenatally irradiated and control rats in the plus maze test Behavioural parameters
Females
Males
Irradiated (n = 9)
Control (n = 9)
Irradiated (n = 9)
Control (n = 9)
Time spent in the open arms (s)
16.5 ± 5.3
32.9 ± 13.9
21.5* ± 9.25
64.9 ± 19.7
Number of rearings
10.7 ± 1.9
9.8 ± 1.5
7.9 ± 0.7
6.2 ± 1.7
7.0 ± 1.8
4.0 ± 1.0
5.1 ± 1.7
9.0 ± 1.8
Number of crossings of the center
Values given as arithmetic means ± SEM. * P < 0.05
Hippocampal neurogenesis We did not find any decrease in proliferation activity in gyrus dentatus of irradiated animals. On the contrary, we have observed even higher number of BrdU-labelled cells in comparison with controls: 514.1 ± 48.44 cells/mm2 (mean ± SEM) in irradiated and 383.17 ± 67.48 cells/ mm2 in control rats, but these differences were not statistically significant. Discussion The effects of ionizing radiation on various forms of innate behaviour and on those of cognitive functions were studied recently. Uma Devi et al. (22) showed that very low doses (0.3–1.5 Gy) of gamma rays applied during sensitive phases of intrauterine development cause learning disorders and memory dysfunctions in mice and the intensity of damage increases with the dose of radiation. Our results indicate relatively mild impairment of short time memory by radiation dose of 1 Gy given on the 16th day of embryonic development, which was expressed in statistically significant increase of the swimming distance needed to find the hidden platform. This effect was significantly more pronounced in females, than in males. Generally, longer swimming distance in females and almost identical time as in males needed to find the platform could be the result of higher prevalence of the tigmotactic Acta Physiologica Hungarica 99, 2012
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behaviour in females, resulting in fast swimming round along the walls of the basin before actively seeking the platform (19). The damage of memory was expressed by a significant prolongation of time needed to find the hidden platform and by the prolongation of the swimming-track in irradiated rats. These results are in agreement with findings of Baskar and Devi (2), who also recorded memory impairment in mice after prenatal irradiation with low doses of gamma-rays. They also studied the effect of radiation on innate forms of behaviour and they found out, that ionizing radiation applied during gravidity evoked disorders of exploratory behaviour: prenatally irradiated mice showed a dose-dependent suppression of locomotory and exploratory activities in the OF and in the light/dark box (light aversion test). Our results contradict to these findings: we recorded an increase in locomotory and exploratory activities in prenatally irradiated rats in the OF and PM. On the other hand, our results correspond well with the findings of Norton and Kimler (15) in rats and with those of Minamisawa and Hirokaga (12) in mice, who also found significant increase in locomotor activity after irradiation with low doses during the sensitive period of gravidity. We suppose that these discrepancies may be due to differences in radiosensitivity among species, strains and genders. Also, the age of animals seems to be an important factor in behavioural response to radiation, being older animals more sensitive, than are younger ones, as it was demonstrated in Fischer-344 rats (15). The gender differences, found in our experiments are in good agreement with data, reporting higher baseline activity levels in OF in female, then in male rats (3, 6). The effects of prenatal irradiation on the emotional state were studied, too. Baskar and Devi (2) noticed significant changes of anxiety in prenatally irradiated mice. Houpert et al. (9) studied the effects of ionizing radiation on the anxiety in rats. Three months before mating, during gestation and during the lactation period they fed the animals with mineral water supplemented with enriched uranium at a dosage of 40 mg 1–1. The progeny of these females, tested in PM showed prolongation of time spent in the open arms of the maze, indicating a decrease of the level of anxiety and an increase of locomotory activity in the OF. We registered an increase of anxiety in irradiated male rats in our experiment, expressed by significantly shortening the time, which the animals spent in the open arms of the maze. The damage of memory functions after irradiation could be related to the changes of hippocampal neurogenesis, which is very sensitive to the ionizing radiation (12). Radiation doses as low as 0.5–1.5 Gy applied on the 14th–17th days of intrauterine development have been found to significantly reduce the number of neurons in various parts of the hippocampus (6, 7, 12). We did not find similar changes in hippocampal neurogenesis. Conversely, we noticed even a mild increase in the number of proliferating cells in gyrus dentatus in irradiated rats in comparison with controls. Similarly, Schmitz et al. (18) did not record statistically significant differences in the total number of neurons in the gyrus dentatus in irradiated rats and Miki et al. (11) did not register any histological changes of the rat’s gyrus dentatus after application of ionizing radiation. We can hypothesize that if prenatal irradiation caused only a partial loss of neural precursor cells this could be compensated by production of new neurons in the process of hippocampal neurogenesis later during the life. Analyses made at different ages could show therefore different stages of this regeneration process of the brain tissue. The results of our study lead us to conclusions, that prenatal irradiation of Wistar rats with a dose of 1 Gy on the 16th day of embryonic development may influence learned as well as innate forms of behaviour in adult age. However, we did not confirm a relation of these changes to the state of hippocampal neurogenesis. Acta Physiologica Hungarica 99, 2012
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Acknowledgement Supported by grant No. 1/0439/10 of agency VEGA of the Slovak Ministry for Education.
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