- Email: [email protected]
Neonatal exposure to a moderate dose of ionizing radiation causes behavioural defects and altered levels of tau protein in mice
NeuroToxicology 45 (2014) 48–55
Contents lists available at ScienceDirect
NeuroToxicology
Neonatal exposure to a moderate dose of ionizing radiation causes behavioural defects and altered levels of tau protein in mice Sonja Buratovic a,*, Bo Stenerlo¨w b, Anders Fredriksson a, Synno¨ve Sundell-Bergman c, Henrik Viberg a, Per Eriksson a a b c
Department of Environmental Toxicology, Uppsala University, Norbyva¨gen 18A, SE-75236 Uppsala, Sweden Department of Radiology, Oncology and Radiation Science, Uppsala University, Uppsala, Sweden Department of Soil and Environment, Swedish University of Agricultural Sciences, Uppsala, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 September 2014 Accepted 18 September 2014 Available online 26 September 2014
Medical use of ionizing radiation (IR) has great benefits for treatment and diagnostic imaging, but procedures as computerized tomography (CT) may deliver a significant radiation dose to the patient. Recently, awareness has been raised about possible non-cancer consequences from low dose exposure to IR during critical phases of perinatal and/or neonatal brain development. In the present study neonatal NMRI mice were whole body irradiated with a single dose of gamma radiation (0; 350 and 500 mGy) on postnatal day 10 (PND 10). At 2 and 4 months of age, mice of both sexes were observed for spontaneous behaviour in a novel home environment. The neuroproteins CaMKII, GAP-43, synaptophysin and total tau in male mouse cerebral cortex and hippocampus were analysed 24 h post-irradiation and in adults at 6 months of age exposed to 0 or 500 mGy on PND 10. A significantly dose-response related deranged spontaneous behaviour in 2- and 4-month-old mice was observed, where both males and females displayed a modified habituation, indicating reduced cognitive function. The dose of 350 mGy seems to be a tentative threshold. Six-month-old male mice showed a significantly increased level of total tau in cerebral cortex after irradiation to 500 mGy compared to controls. This demonstrates that a single moderate dose of IR, given during a defined critical period of brain development, is sufficient to cause persistently reduced cognitive function. Moreover, an elevation of tau protein was observed in male mice displaying reduced cognitive function. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Neonatal Brain development Ionizing radiation Behaviour Tau
1. Introduction The use of ionizing radiation (IR) in treatment and medical diagnostic procedures has increased over the past decade and is now the major artificial source of IR exposure (Bernier et al., 2012). Although computerized tomography (CT) scans only make up a fraction of all X-ray examinations annually performed, it has come to represent 40–67% of the received medical radiation dose in the population (Mettler et al., 2000; Bernier et al., 2012) A British epidemiological study estimated the absorbed dose of a single brain CT for children under the age of 10 years, in 2001–2008, to be approximately 30 mGy (Pearce et al., 2012b). In the USA, between the years of 1998 and 1999, children below the age of 15 were subjected to approximately 11% of all performed CT scans, of which around 50% were directed towards the cranial area. CT scans of the
* Corresponding author. Tel.: +46 184717699. E-mail address: [email protected] (S. Buratovic). http://dx.doi.org/10.1016/j.neuro.2014.09.002 0161-813X/ß 2014 Elsevier Inc. All rights reserved.
head region contributed 13.9% of the total effective dose in diagnostic radiology with an average effective dose of 1.5 mSv/ procedure (Mettler et al., 2000). Although much attention has been focused on potential late cancer risk following exposure to IR in children for treatment of brain tumours, exposure at very young age may also influence the development of the central nervous system (CNS). An epidemiological study indicated that exposure to IR, during early human development can have a negative impact on cognitive development during childhood (Hall et al., 2004). In many mammalian species the newborn period coincides with a period of rapid growth and development of the brain, the ‘brain growth spurt’ (BGS) (Davison and Dobbing, 1968). In humans, the ‘BGS’ begins during the third trimester of gestation and continues throughout the first 2 years of ex utero life. In mouse and rat this period is neonatal, spanning the first 3–4 weeks of life, during which the brain undergoes several fundamental developmental phases, viz. axonal and dendritic outgrowth, the establishment of neural connections, acquisition of new sensory and motor faculties (Bolles and Woods, 1964), resulting in a peak in
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
spontaneous behaviour, and numerous biochemical changes that transform the feto-neonatal brain into that of the mature adult (Davison and Dobbing, 1968; Campbell et al., 1969; Abreu-Villaca et al., 2011). During this period of rapid brain development in mice, a distinct ontogeny of certain neuroproteins can be observed (Viberg et al., 2008; Viberg, 2009). CaMKII is known to be one of the most abundant protein kinases and is believed to play a crucial role in dendritic arborisation, long-term potentiation, memory and learning (Erondu and Kennedy, 1985; Lisman et al., 2002; Yamauchi, 2005). Growth-associated protein 43 (GAP-43) is most commonly found in the growth cone of axons and is also believed to be important for long term potentiation (Benowitz and Routtenberg, 1997; Oestreicher et al., 1997). Synaptophysin is believed to be involved in neuronal plasticity by regulating cycling and formation of synaptic vesicles (Sarnat and Born, 1999). Tau is a member of the microtubule-associated protein family which functions to stabilize and maintain a normal morphology of neurons, establish polarity and support the outgrowth of neural processes (Wang and Liu, 2008). Elevated levels of the phosphorylated tau isoform have been observed to impair normal memory and learning functions in humans and it is therefore used as a diagnostic marker in the clinic for diagnosing Alzheimer’s disease. In a previous study irradiation of neonatal mice to a single dose of 500 mGy was shown to induce persistent alterations in adult male mouse spontaneous behaviour as well as a reduced memory and learning capacity, when the exposure occurred during a critical period of the BGS (Eriksson et al., 2010). The present study was conducted to investigate the effect after neonatal exposure to a single moderate dose of gamma radiation on (1) spontaneous behaviour and habituation to a novel home environment in adult male and female mice and (2) levels of important neuroproteins in cerebral cortex and hippocampus of neonatal and adult male mice.
49
homogeneous 3%, over the 10 cm in diameter large dish area. Control mice were placed in the same plastic dishes as the irradiated mice and sham irradiated. Animals were not anesthetized during irradiation. Each exposure group comprised mice from 3 to 4 different litters. 2.3. Spontaneous behaviour Spontaneous behaviour, in a novel home environment, was observed in mice of both sexes at 2- and 4-months of age. The observations and recordings were carried out between 08.00 and 13.00 h, under the same light and temperature conditions as their housing conditions. During 60 consecutive minutes an automated system recorded the motor activity of the animals, and recordings of the variables locomotion, rearing and total activity were made (Rat-O-Matic, ADEA Electronik AB, Uppsala, Sweden) as described by Fredriksson (1994). Twelve cages, placed in individual soundproof boxes with separate ventilation were used. Locomotion: Movements made in the horizontal plane were registered by the low level (10 mm above the bedding material) infrared beams. Rearing: Movements made in the vertical plane were registered by the high level (80 mm above the bedding material) infrared beam. Total activity: A needle mounted on a horizontal arm with a counterweight connected to the test cage registered all vibrations such as movements, grooming and shaking.
2. Materials and methods
All data were collected electronically through a computer interface. The animals were observed for a 60 min period of time (0–60 min) divided into three 20 min intervals; in each 60-min session animals from each exposure group were represented. A total of 12 males and 12 females per exposure group, where 3–4 individuals were taken randomly from at least 3 different litters, were observed for spontaneous behaviour.
2.1. Animals
2.4. Slot Blot analysis
Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC), after approval from the local ethical committees (Uppsala University and the Agricultural Research Council) and by the Swedish Committee for Ethical Experiments on Laboratory Animals. Pregnant Naval Medical Research Institute (NMRI) mice were purchased from Scanbur, Sollentuna, Sweden. The animals were housed individually in plastic cages in a room with an ambient temperature of 22 8C and a 12/12 h constant light/dark cycle. Animals were supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. Females were checked for birth twice daily (08.00 and 18.00 h) and day of birth was designated day 0. Within the first 48 h after birth, litter sizes were adjusted to 10–12 pups of both sexes by euthanizing excess pups. At approximately 4 weeks of age, male and female offspring were separated with regard to sex and raised in sibling groups of 3–7 individuals in separate male and female rooms.
Male mice irradiated to a dose of 500 mGy were used in the Slot Blot analysis as they showed an altered spontaneous behaviour and lack of habituation. Sham irradiated mice were used as controls. The mice were sacrificed by cervical dislocation 24 h post-irradiation or at 6 months of age. The brains were dissected on an ice-cold glass plate. Cerebral cortex and hippocampus were collected, snap frozen in liquid nitrogen and stored at 80 8C until assayed. Both brain regions were homogenized in a RIPA cell lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 20 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% sodium deoxycholate) to which a 0.5% protease inhibitor cocktail (Protease Inhibitor Cocktail set III, Calbiochem) was added. Homogenates were centrifuged at 14,000 g at 4 8C for 15 min and supernatant analyzed for protein content by using the BCA method (Pierce). Homogenates were stored at 80 8C. Viberg and co-workers have previously evaluated the specificity of antibodies CaMKII (Upstate Millpore, 05-552), GAP-43 (Chemicon Millipore, AB5220), synaptophysin (Calbiochem, 573822) and tau (Santa Cruz, 32274) by Western blot procedure with satisfactory results (Viberg et al., 2008; Viberg, 2009). The antibody used against tau recognizes both the nonphosphorylated and phosphorylated protein forms. Four mg of protein for CaMKII and GAP-43, 3 mg for synaptophysin and 3.5 mg for tau were diluted in sample buffer (120 mM KCl, 20 mM NaCl, 2 mM NaHCO3, 2 mM MgCl2, 5 mM HEPES, pH 7.4, 0.05% Tween20, 0.2% NaN3) to a final volume of 200 ml. Duplicates of each sample were applied to a nitrocellulose membrane (0.45 mm,
2.2. Irradiation Mice of both sexes were whole body gamma-irradiated on postnatal day 10 to a single dose from a 60Co source at The Svedberg laboratory, Uppsala University, Uppsala, Sweden (Eriksson et al., 2010). Mice were placed in plastic dishes and exposed to a single dose of 350 or 500 mGy with a surface dose rate of about 0.02 Gy/minute. An ionization chamber (Markus chamber type 23343, PTW-Freiburg) was used to measure the dose, which was
50
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
BioRad) using a Bio-Dot SF microfiltration apparatus (BioRad). Membranes were fixed in 25% isopropanol and 10% acetic acid solution for 15 min, washed and subsequently blocked for 1 h in 5% non-fat dry milk and 0.03% Tween-20 in room temperature. Incubation of membranes with primary mouse monoclonal CaMKII antibody (1:5000), rabbit polyclonal GAP-43 antibody (1:10,000), mouse monoclonal synaptophysin antibody (1:10,000) or mouse monoclonal tau antibody (1:1000) occurred overnight at 4 8C. A horseradish peroxidase conjugate secondary antibody against mouse (074-1806, 1:20,000) or rabbit (KPL 074-1506) was used to detect immunoreactivity. Immunoreactive bands were traced using an enhanced chemiluminescent substrate (Pierce, Super Signal West Dura) and imaged on a LAS-1000 (Fuji Film, Tokyo, Japan). Band intensity was quantified using IR-LAS 1000 Pro (Fuji Film). 2.5. Statistical analysis Data from the variables locomotion, rearing and total activity over the three consecutive 20-min time periods (treatment, time and treatment time, between subjects, within subjects and interaction factors, respectively) in the spontaneous behaviour in a novel home environment were submitted to a split-plot ANOVA design and pairwise testing using a Duncan’s MRT (multiple range test) test in SAS 9.1 software (Kirk, 1968; Festing, 2006; Eriksson, 2008; Lazic and Essioux, 2013). Evaluation of the Slot Blot analyses of CaMKII, GAP-43, synaptophysin and tau in cerebral cortex and hippocampus from male mice was made using one-way ANOVA, pairwise testing Duncan’s MRT (STATISTICA 10).
3. Results No overt visual signs of toxicity were observed in the irradiated mice throughout the experimental period. Nor were there any significant deviations in body weight between irradiated mice and control mice (data not shown). 3.1. Spontaneous behaviour in 2-month-old male mice The spontaneous behaviour variables locomotion, rearing and total activity in 2-month-old male mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 1. Significant treatment time interactions were observed (F4,66 = 220.47; F4,66 = 316.25; F4,66 = 144.45, p 0.001) for locomotion, rearing and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three test variables. Control males showed a normal decrease in activity over the 60 min period of testing time, as the novelty of the test chamber diminished, and habituated in a normal way, as earlier reported by Eriksson and co-workers (2010) and Fredriksson (1994). Male mice exposed to 350 mGy showed a significantly (p 0.01) decreased activity in the variables locomotion, rearing, and total activity during the first 20-min period, compared to control animals. Mice exposed to 500 mGy showed significantly (p 0.01) decreased locomotion, rearing and total activity during the first 20 min of testing, compared with control animals. Mice exposed to 500 mGy showed significantly (p 0.01) decreased locomotion and total activity during the first 20 min of testing, compared with animals exposed to 350 mGy. During the last 20 min of testing, mice exposed to 500 mGy showed significantly (p 0.01) increased locomotion, rearing, and total activity, compared to controls and animals exposed to 350 mGy (Fig. 1).
Fig. 1. Spontaneous behaviour of 2-month-old NMRI male mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 220.47; F4,66 = 316.25; F4,66 = 144.45) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (a) significantly different vs. control, p 0.05; (B) significantly different vs. 350 mGy, p 0.01. Height of bars represents mean SD.
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
51
3.2. Spontaneous behaviour in 2-month-old female mice The spontaneous behaviour variables locomotion, rearing, and total activity in 2-month-old female mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 2. Significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 134.87; F4,66 = 72.81, p 0.001) for locomotion, rearing, and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three-test variables. Control female mice showed normal decrease in activity and habituation capacity over the 60 min period of testing time, as seen in age-matched male mice. Females exposed to 350 mGy showed a significantly (p 0.01) decreased activity in the variables locomotion, rearing, and total activity during the first 20 min period, compared to control animals. Females exposed to 500 mGy showed significantly (p 0.01) decreased locomotion, rearing and total activity during the first 20 min of testing, compared with control females. Mice exposed to 500 mGy showed significantly (p 0.01) decreased locomotion and total activity during the first 20 min of testing, compared with females exposed to 350 mGy. During the last 20 min of testing, females exposed to 500 mGy showed significantly (p 0.01) increased locomotion, rearing, and total activity, compared to female controls and females exposed to 350 mGy (Fig. 2). 3.3. Spontaneous behaviour in 4-month-old male mice The spontaneous behaviour variables locomotion, rearing and total activity in 4-month-old male mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 3. Significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 1334.87; F4,66 = 72.8, p 0.01) for locomotion, rearing and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three test variables. Control males showed a normal decrease in activity and habituation capacity over the 60 min period of testing time, as was observed in 2-month-old male mice. Alterations in spontaneous behaviour observed at 4 months of age, following exposure to 350 mGy or 500 mGy, was in concordance with observed spontaneous behaviour alterations at 2 months of age and no additional significant changes were observed. 3.4. Spontaneous behaviour in 4-month-old female mice The spontaneous behaviour variables locomotion, rearing and total activity in 4-month-old female mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 4. Significant treatment time interactions were observed (F4,66 = 53.21; F4,66 = 93.68; F4,66 = 42.33, p 0.01) for locomotion, rearing and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three test variables. Control females showed a normal decrease in activity and habituation capacity over the 60 min period of testing time, as was observed in age-matched male mice and in 2-month-old females. Alterations in spontaneous behaviour observed in 4-month-old females, following exposure to 350 mGy or 500 mGy, was in concordance with observed spontaneous behaviour alterations in 2-month-old female mice and also age-matched males, with no additional significant changes being observed.
Fig. 2. Spontaneous behaviour of 2-month-old NMRI female mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 134.87; F4,66 = 72.81) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (B) significantly different vs. 350 mGy, p 0.01. Height of bars represents mean SD.
52
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
Fig. 3. Spontaneous behaviour of 4-month-old NMRI male mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 1334.87; F4,66 = 72.81) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (a) significantly different vs. control, p 0.05; (B) significantly different vs. 350 mGy, p 0.01. Height of bars represents mean SD.
Fig. 4. Spontaneous behaviour of 4-month-old NMRI female mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 53.21; F4,66 = 93.68; F4,66 = 42.33) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (a) significantly different vs. control, p 0.05; (B) significantly different vs. 350 mGy, p 0.01; (b) significantly different vs. 350 mGy, p 0.05. Height of bars represents mean SD.
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
53
Table 1 Protein levels of CaMKII, GAP-43, synaptophysin and tau in male mice exposed to 500 mGy on postnatal day 10.a 11-day old
Control CaMKII GAP-43 Synaptophysin Tau
6-month-old
Cerebral cortex
Hippocampus
Cerebral cortex
Hippocampus
100 124 18 108 6 154 19** 218 14***
100 102 15 94 8 109 13 67 14**
100 114 24 100 10 114 10 205 17***
100 105 9 98 8 106 9 106 8
a Animals were sacrificed as neonatals 24 h post-irradiation or at the adult age of 6 months. Cerebral cortex and hippocampus were harvested and protein levels analysed using the Slot-Blot technique. The data were subjected to a one-way ANOVA, pairwise testing between control and 500 Gy exposed mice using Duncan’s test. The control value was set to 100% (SD) and the statistical difference between control and 500 Gy exposed mice is indicated with ** for p 0.01 or *** for p 0.001.
3.5. Analysis of levels of CaMKII, GAP-43 synaptophysin and tau in the cerebral cortex and hippocampus of 11-day-old and 6-month-old male mice At 11 days: Brain homogenates from hippocampus and cerebral cortex, collected 24 h post-irradiation, from male mice exposed on PND 10 to 0 or 500 mGy were analysed for levels of CaMKII, GAP-43, synaptophysin and total tau using the Slot-Blot technique. The results are presented in Table 1 as the percentage of control animal protein levels. Protein levels of sham irradiated animals were normalized to 100%. Significant (p 0.001) increase (118%) in tau levels as well as significant (p 0.01) increase (54%) in levels of synaptophysin were observed in cerebral cortex in male mice exposed to 500 mGy. Hippocampal levels of tau were significantly (p 0.01) decreased (33%) in male mice exposed to 500 mGy on PND 10, when compared to controls. At 6 months: Brain homogenates from cerebral cortex and hippocampus prepared from 6-month-old male mice, exposed on PND 10 to 0 or 500 mGy, were analysed for levels of the same neuroproteins as given above. The results are presented in Table 1 as the percentage of sham irradiated control animal protein levels, which were standardized to 100%. Six-month-old male mice, exposed to 500 mGy on PND 10, expressed significantly (p 0.001) increased (105%) levels of tau protein in cerebral cortex. No significant changes in neuroprotein levels were seen in the hippocampus (Table 1). 4. Discussion The present study shows that neonatal radiation exposure can cause similar persistent neurobehavioural deficits in male and female mice and that a threshold dose is seen around 350 mGy. Furthermore, in adult male mice showing reduced cognitive function, the level of total tau was increased in the cerebral cortex. An alteration in neuroprotein levels was also observed in neonatal mouse cerebral cortex and hippocampus following the whole body external irradiation. Male mice neonatally exposed to doses of 350 and 500 mGy displayed significantly aberrant spontaneous behaviour and a dose-response related decrease in habituation at the age of 2 and 4 months. During the spontaneous behaviour observation a decrease in sensoric stimuli, following the diminishment of the novelty of the test chamber rendered the control animals to respond less to these stimuli by displaying less activity and habituating to the novel home environment. Therefore, habituation can be used a measurement of cognitive function (Daenen et al., 2001; Rankin et al., 2009). Habituation, defined here as a decrease in the variables locomotion, rearing and total activity counts, was evident in control animals whereas mice irradiated with 500 mGy displayed reduced activity early in the 60 min test period, but a hyperactive behaviour toward the end. This
aberrant spontaneous behaviour in adult male mice following neonatal exposure to gamma radiation is in agreement with our earlier reported study (Eriksson et al., 2010). In that study a single dose of 500 mGy resulted in a loss of habituation to a novel home environment, accompanied by a hyperactive behaviour at the end of the observational period, while no effect was seen after a single dose of 200 mGy. In the present study a modified habituation was observed at 350 mGy, where a significant decrease in activity during the first 20-min period of testing indicates 350 mGy as a possible threshold value for induction of persistent neurobehavioural defects. Developmental exposure to radiation in studies by Hossain and Devi (2000, 2001) indicated persistent changes in adult mouse locomotor activity in the open-field test as well as memory and learning impairments following exposure to 500 mGy irradiation during the embryonic period of brain development (Hossain and Devi, 2000, 2001). Multiple studies conducted to investigate neurotoxic effects following internal exposure to uranium in adult animals have shown that exposure to depleted uranium causes alterations in sleep-wake cycles as well as food intake, while exposure to enriched uranium resulted in additional alterations in sleeping patterns as well as impairments in spatial working memory and higher levels of anxiety-like behaviour in rats (Houpert et al., 2005; Lestaevel et al., 2005a, 2005b). Houpert and co-workers (2005) suggest that the radiation from enriched uranium is the main inducer of the observed disruptions in working memory and anxiety-like behaviour since exposure to depleted uranium for an equivalent time period did not result in similar behavioural alterations. In female mice the spontaneous behaviour test also revealed a dose-response related disruption of habituation capacity in both 2-month-old and 4-month-old mice following neonatal irradiation to 350 or 500 mGy. This effect was similar to the altered habituation observed in the age-matched male mice. This shows that female mice can be as susceptible as male mice to develop aberrant spontaneous behaviour and loss of habituation to a novel home environment following a single neonatal exposure to radiation. Furthermore, this indicates that there are no major differences between sexes regarding manifestation of this type of developmental neurotoxic defects in mice exposed to radiation. Other studies suggest that female mice, acutely irradiated with 8 Gy while under anaesthesia on PND 14, develop more pronounced manifestations of impaired white matter growth, impaired hippocampal neurogenesis and anxiety at an adult age when compared to males (Roughton et al., 2012, 2013). One cannot rule out a possible interaction effect between radiation and the anaesthetic as a causative factor for the sex differences observed by Roughton and co-workers. Whether there are sex differences in mechanisms of induction of neurotoxicity of high dose exposure compared to low dose exposure to radiation remains to be investigated.
54
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
During the BGS a distinct ontogeny of several important neuroproteins such as CaMKII, GAP-43, synaptophysin and tau can be observed, where the most pronounced increase in proteins occurs around PND 7–14 (Viberg et al., 2008; Viberg, 2009). The present study show elevated levels of synaptophysin and tau in cerebral cortex of neonatal male mice exposed to a single dose of 500 mGy, but also a decrease in hippocampal total tau levels. Changes in levels of neuroproteins have been reported following adult exposure to radiation. A reduction in CaMKII levels in adult male mice, but not female was observed, following 500 mGy X-ray irradiation at the adult age of 45 day (Silasi et al., 2004). Interestingly, Goutan and co-workers (1999) found a decrease in cerebellar GAP-43 levels but no alterations in synaptophysin were observed following an acute 2 Gy whole body gamma radiation dose on PND 1. This alteration was not present 48 h postirradiation indicating a capability of the cerebellum to recover after irradiation during postnatal development (Goutan et al., 1999). Keeping in mind the distinct ontogeny of CaMKII, GAP-43, synaptophysin and tau during the BGS, it is reasonable to assume that disruptions in protein levels and the ability of the brain to recover from insults may vary depending at which time-point of brain development the exposure occurs (Viberg et al., 2008; Viberg, 2009). A defined critical period, around PND 10 in mouse, for induction of persistent behavioural defects, including altered cognitive function and changes in cholinergic receptors, has been seen after exposure to different types of chemicals (Eriksson et al., 1992, 2000; Eriksson, 1997). It is worth noting, that the elevated level of tau protein observed in cerebral cortex of neonatal male mice is still present in 6-month-old animals. Alzheimer’s disease (AD) is characterized by a progressive and profound loss of cognitive functions presumably attributed to cholinergic system dysfunction in early stages and the presence of intracellular neurofibrillary tangles consisting of hyper-phosphorylated aggregates of tau proteins in later stages of disease progression (Francis et al., 1999; Hardy and Selkoe, 2002; Di Domenico et al., 2011). An elevated level of phosphorylated tau protein in cerebrospinal fluid is used in medicine as a diagnostic marker for AD (Ikeda et al., 2013). However, in this study alterations in neuroproteins and behaviour were prominently present at higher radiation doses (500 mGy) than received during a single CT scan, which has been estimated to deliver an average dose of 30 mGy to the brain (Pearce et al., 2012b). Still it is important to consider possible late neurocognitive defects deriving from irradiation of non-target tissue in radiotherapy of brain tumours in childhood (Mulhern et al., 2004), as well as repeated CT scans in children. It has been estimated that approximately 30% of patients younger than 22 years old underwent more than one CT scan in Great Britain during the years 1993–2002 (Pearce et al., 2012a). Taking into consideration that the radiation dose delivered to the brain during a single CT scan can vary from 21 to 153 mGy, according to a recent Swedish report form the Radiation Safety Authority (Leitz and Alme´n, 2010), the tentative threshold dose (350 mGy) observed to induce spontaneous behaviour alterations in this study is well within the range of a plausible exposure situation. 5. Conclusion The present study shows that irradiation to a single dose of 500 mGy can cause developmental neurotoxic effects, similar in both male and female mice, manifested as a lack of or reduced capacity to habituate to a novel home environment. Moreover, irradiation to a dose of 350 mGy seems to be a tentative threshold for induction of this type of neurotoxicity. Neonatal exposure to 500 mGy caused increased levels of tau protein in the cerebral cortex of neonatal male mice. This trait was also observable in
adult male mice, showing reduced cognitive function. The finding of increased levels of total tau protein in mice calls for further investigation, as phosphorylated tau is a diagnostic marker in humans with AD. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This work was supported by the European Community’s Seventh Framework Programme (EURATOM) [CEREBRAD; grant number 29552] and the Swedish Radiation Safety Authority. References Abreu-Villaca Y, Filgueiras CC, Manhaes AC. Developmental aspects of the cholinergic system. Behav Brain Res 2011;221:367–78. Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci 1997;20:84–91. Bernier MO, Rehel JL, Brisse HJ, Wu-Zhou X, Caer-Lorho S, Jacob S, et al. Radiation exposure from CT in early childhood: a French large-scale multicentre study. Br J Radiol 2012;85:53–60. Bolles RC, Woods PJ. The ontogeny of behaviour in the albino rat. Anim Behav 1964;12: 427–41. Campbell BA, Lytle LD, Fibiger HC. Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science 1969;166:635–7. Daenen EWPM, Van der Heyden JA, Kruse CG, Wolterink G, Van Ree JM. Adaptation and habituation to an open field and responses to various stressful events in animals with neonatal lesions in the amygdala or ventral hippocampus. Brain Res 2001;918: 153–65. Davison AN, Dobbing J. Applied neurochemistry. Oxford: Blackwell; 1968. Di Domenico F, Sultana R, Barone E, Perluigi M, Cini C, Mancuso C, et al. Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer’s disease subjects. J Proteomics 2011;74:1091–103. Eriksson P. Developmental neurotoxicity of environmental agents in the neonate. Neurotoxicology 1997;18:719–26. Eriksson P. Response to: use of the pup as the statistical unit in developmental neurotoxicity studies: overlooked model or poor research design? Toxicol Sci 2008;103:411–3. Eriksson P, Ahlbom J, Fredriksson A. Exposure to DDT during a defined period in neonatal life induces permanent changes in brain muscarinic receptors and behaviour in adult mice. Brain Res 1992;582:277–81. Eriksson P, Ankarberg E, Fredriksson A. Exposure to nicotine during a defined period in neonatal life induces permanent changes in brain nicotinic receptors and in behaviour of adult mice. Brain Res 2000;853:41–8. Eriksson P, Fischer C, Stenerlow B, Fredriksson A, Sundell-Bergman S. Interaction of gamma-radiation and methyl mercury during a critical phase of neonatal brain development in mice exacerbates developmental neurobehavioural effects. Neurotoxicology 2010;31:223–9. Erondu NE, Kennedy MB. Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain. J Neurosci 1985;5:3270–7. Festing MFW. Design and statistical methods in studies using animal models of development. Ilar J 2006;47:5–14. Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic hypothesis of Alzheimer’s disease: a review of progress. J Neurol Neurosurg Psychiatry 1999;66:137–47. Fredriksson A. MPTP-induced behavioural deficits in mice: validity and utility of a model of parkinsonism. Uppsala: Uppsala University; 1994. Goutan E, Marti E, Ferrer I. Expression of synaptic proteins in the developing rat cerebellum following ionizing radiation. Int J Dev Neurosci 1999;17:275–83. Hall P, Adami HO, Trichopoulos D, Pedersen NL, Lagiou P, Ekbom A, et al. Effect of low doses of ionising radiation in infancy on cognitive function in adulthood: Swedish population based cohort study. Br Med J 2004;328:19. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353–6. Hossain M, Devi PU. Effect of irradiation at the early fetal stage on adult brain function in the mouse: locomotor activity. Int J Radiat Biol 2000;76:1397–402. Hossain M, Devi PU. Effect of irradiation at the early foetal stage on adult brain function of mouse: learning and memory. Int J Radiat Biol 2001;77:581–5. Houpert P, Lestaevel P, Bussy C, Paquet F, Gourmelon P. Enriched but not depleted uranium affects central nervous system in long-term exposed rat. Neurotoxicology 2005;26:1015–20. Ikeda M, Yonemura K, Kakuda S, Tashiro Y, Fujita Y, Takai E, et al. Cerebrospinal fluid levels of phosphorylated tau and Abeta1–38/Abeta1–40/Abeta1–42 in Alzheimer’s disease with PS1 mutations. Amyloid 2013;20:107–12. Kirk RE. Procedures for the behavioural science. Belmont, CA: Brooks/Cole; 1968. Lazic SE, Essioux L. Improving basic and translational science by accounting for litterto-litter variation in animal models. BMC Neurosci 2013;14:37.
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55 Leitz W, Alme´n A. Patientdoser fra˚n ro¨ntgenunderso¨kningar i Sverige – utveckling fra˚n 2005 till 2008. Swedish Radiation Safety Authority; 2010. Lestaevel P, Bussy C, Paquet F, Dhieux B, Clarencon D, Houpert P, et al. Changes in sleepwake cycle after chronic exposure to uranium in rats. Neurotoxicol Teratol 2005a;27:835–40. Lestaevel P, Houpert P, Bussy C, Dhieux B, Gourmelon P, Paquet F. The brain is a target organ after acute exposure to depleted uranium. Toxicology 2005b;212:219–26. Lisman J, Schulman H, Cline H. The molecular basis of CaMKII function in synaptic and behavioural memory. Nat Rev 2002;3:175–90. Mettler FA Jr, Wiest PW, Locken JA, Kelsey CA. CT scanning: patterns of use and dose. J Radiol Prot 2000;20:353–9. Mulhern RK, Merchant TE, Gajjar A, Reddick WE, Kun LE. Late neurocognitive sequelae in survivors of brain tumours in childhood. Lancet Oncol 2004;5:399–408. Oestreicher AB, De Graan PN, Gispen WH, Verhaagen J, Schrama LH. B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system. Prog Neurobiol 1997;53:627–86. Pearce MS, Salotti JA, Howe NL, McHugh K, Kim KP, Lee C, et al. CT scans in young people in Great Britain: temporal and descriptive patterns, 1993–2002. Radiol Res Pract 2012;594278. Pearce MS, Salotti JA, Little MP, McHugh K, Lee C, Kim KP, et al. Radiation exposure from CT scans in childhood and subsequent risk of leukaemia and brain tumours: a retrospective cohort study. Lancet 2012b;380:499–505. Rankin CH, Abrams T, Barry RJ, Bhatnagar S, Clayton DF, Colombo J, et al. Habituation revisited: an updated and revised description of the behavioral characteristics of habituation. Neurobiol Learn Mem 2009;92:135–8.
55
Roughton K, Bostrom M, Kalm M, Blomgren K. Irradiation to the young mouse brain impaired white matter growth more in females than in males. Cell Death Dis 2013;4:e897. Roughton K, Kalm M, Blomgren K. Sex-dependent differences in behavior and hippocampal neurogenesis after irradiation to the young mouse brain. Eur J Neurosci 2012;36:2763–72. Sarnat HB, Born DE. Synaptophysin immunocytochemistry with thermal intensification: a marker of terminal axonal maturation in the human fetal nervous system. Brain Dev 1999;21:41–50. Silasi G, Diaz-Heijtz R, Besplug J, Rodriguez-Juarez R, Titov V, Kolb B, et al. Selective brain responses to acute and chronic low-dose X-ray irradiation in males and females. Biochem Biophys Res Commun 2004;325:1223–35. Wang JZ, Liu F. Microtubule-associated protein tau in development, degeneration and protection of neurons. Prog Neurobiol 2008;85:148–75. Viberg H. Neonatal ontogeny and neurotoxic effect of decabrominated diphenyl ether (PBDE 209) on levels of synaptophysin and tau. Int J Dev Neurosci 2009;27: 423–9. Viberg H, Mundy W, Eriksson P. Neonatal exposure to decabrominated diphenyl ether (PBDE 209) results in changes in BDNF, CaMKII and GAP-43, biochemical substrates of neuronal survival, growth, and synaptogenesis. Neurotoxicology 2008;29: 152–9. Yamauchi T. Neuronal Ca2+/calmodulin-dependent protein kinase II – discovery, progress in a quarter of a century, and perspective: implication for learning and memory. Biol Pharm Bull 2005;28:1342–54.
Contents lists available at ScienceDirect
NeuroToxicology
Neonatal exposure to a moderate dose of ionizing radiation causes behavioural defects and altered levels of tau protein in mice Sonja Buratovic a,*, Bo Stenerlo¨w b, Anders Fredriksson a, Synno¨ve Sundell-Bergman c, Henrik Viberg a, Per Eriksson a a b c
Department of Environmental Toxicology, Uppsala University, Norbyva¨gen 18A, SE-75236 Uppsala, Sweden Department of Radiology, Oncology and Radiation Science, Uppsala University, Uppsala, Sweden Department of Soil and Environment, Swedish University of Agricultural Sciences, Uppsala, Sweden
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 September 2014 Accepted 18 September 2014 Available online 26 September 2014
Medical use of ionizing radiation (IR) has great benefits for treatment and diagnostic imaging, but procedures as computerized tomography (CT) may deliver a significant radiation dose to the patient. Recently, awareness has been raised about possible non-cancer consequences from low dose exposure to IR during critical phases of perinatal and/or neonatal brain development. In the present study neonatal NMRI mice were whole body irradiated with a single dose of gamma radiation (0; 350 and 500 mGy) on postnatal day 10 (PND 10). At 2 and 4 months of age, mice of both sexes were observed for spontaneous behaviour in a novel home environment. The neuroproteins CaMKII, GAP-43, synaptophysin and total tau in male mouse cerebral cortex and hippocampus were analysed 24 h post-irradiation and in adults at 6 months of age exposed to 0 or 500 mGy on PND 10. A significantly dose-response related deranged spontaneous behaviour in 2- and 4-month-old mice was observed, where both males and females displayed a modified habituation, indicating reduced cognitive function. The dose of 350 mGy seems to be a tentative threshold. Six-month-old male mice showed a significantly increased level of total tau in cerebral cortex after irradiation to 500 mGy compared to controls. This demonstrates that a single moderate dose of IR, given during a defined critical period of brain development, is sufficient to cause persistently reduced cognitive function. Moreover, an elevation of tau protein was observed in male mice displaying reduced cognitive function. ß 2014 Elsevier Inc. All rights reserved.
Keywords: Neonatal Brain development Ionizing radiation Behaviour Tau
1. Introduction The use of ionizing radiation (IR) in treatment and medical diagnostic procedures has increased over the past decade and is now the major artificial source of IR exposure (Bernier et al., 2012). Although computerized tomography (CT) scans only make up a fraction of all X-ray examinations annually performed, it has come to represent 40–67% of the received medical radiation dose in the population (Mettler et al., 2000; Bernier et al., 2012) A British epidemiological study estimated the absorbed dose of a single brain CT for children under the age of 10 years, in 2001–2008, to be approximately 30 mGy (Pearce et al., 2012b). In the USA, between the years of 1998 and 1999, children below the age of 15 were subjected to approximately 11% of all performed CT scans, of which around 50% were directed towards the cranial area. CT scans of the
* Corresponding author. Tel.: +46 184717699. E-mail address: [email protected] (S. Buratovic). http://dx.doi.org/10.1016/j.neuro.2014.09.002 0161-813X/ß 2014 Elsevier Inc. All rights reserved.
head region contributed 13.9% of the total effective dose in diagnostic radiology with an average effective dose of 1.5 mSv/ procedure (Mettler et al., 2000). Although much attention has been focused on potential late cancer risk following exposure to IR in children for treatment of brain tumours, exposure at very young age may also influence the development of the central nervous system (CNS). An epidemiological study indicated that exposure to IR, during early human development can have a negative impact on cognitive development during childhood (Hall et al., 2004). In many mammalian species the newborn period coincides with a period of rapid growth and development of the brain, the ‘brain growth spurt’ (BGS) (Davison and Dobbing, 1968). In humans, the ‘BGS’ begins during the third trimester of gestation and continues throughout the first 2 years of ex utero life. In mouse and rat this period is neonatal, spanning the first 3–4 weeks of life, during which the brain undergoes several fundamental developmental phases, viz. axonal and dendritic outgrowth, the establishment of neural connections, acquisition of new sensory and motor faculties (Bolles and Woods, 1964), resulting in a peak in
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
spontaneous behaviour, and numerous biochemical changes that transform the feto-neonatal brain into that of the mature adult (Davison and Dobbing, 1968; Campbell et al., 1969; Abreu-Villaca et al., 2011). During this period of rapid brain development in mice, a distinct ontogeny of certain neuroproteins can be observed (Viberg et al., 2008; Viberg, 2009). CaMKII is known to be one of the most abundant protein kinases and is believed to play a crucial role in dendritic arborisation, long-term potentiation, memory and learning (Erondu and Kennedy, 1985; Lisman et al., 2002; Yamauchi, 2005). Growth-associated protein 43 (GAP-43) is most commonly found in the growth cone of axons and is also believed to be important for long term potentiation (Benowitz and Routtenberg, 1997; Oestreicher et al., 1997). Synaptophysin is believed to be involved in neuronal plasticity by regulating cycling and formation of synaptic vesicles (Sarnat and Born, 1999). Tau is a member of the microtubule-associated protein family which functions to stabilize and maintain a normal morphology of neurons, establish polarity and support the outgrowth of neural processes (Wang and Liu, 2008). Elevated levels of the phosphorylated tau isoform have been observed to impair normal memory and learning functions in humans and it is therefore used as a diagnostic marker in the clinic for diagnosing Alzheimer’s disease. In a previous study irradiation of neonatal mice to a single dose of 500 mGy was shown to induce persistent alterations in adult male mouse spontaneous behaviour as well as a reduced memory and learning capacity, when the exposure occurred during a critical period of the BGS (Eriksson et al., 2010). The present study was conducted to investigate the effect after neonatal exposure to a single moderate dose of gamma radiation on (1) spontaneous behaviour and habituation to a novel home environment in adult male and female mice and (2) levels of important neuroproteins in cerebral cortex and hippocampus of neonatal and adult male mice.
49
homogeneous 3%, over the 10 cm in diameter large dish area. Control mice were placed in the same plastic dishes as the irradiated mice and sham irradiated. Animals were not anesthetized during irradiation. Each exposure group comprised mice from 3 to 4 different litters. 2.3. Spontaneous behaviour Spontaneous behaviour, in a novel home environment, was observed in mice of both sexes at 2- and 4-months of age. The observations and recordings were carried out between 08.00 and 13.00 h, under the same light and temperature conditions as their housing conditions. During 60 consecutive minutes an automated system recorded the motor activity of the animals, and recordings of the variables locomotion, rearing and total activity were made (Rat-O-Matic, ADEA Electronik AB, Uppsala, Sweden) as described by Fredriksson (1994). Twelve cages, placed in individual soundproof boxes with separate ventilation were used. Locomotion: Movements made in the horizontal plane were registered by the low level (10 mm above the bedding material) infrared beams. Rearing: Movements made in the vertical plane were registered by the high level (80 mm above the bedding material) infrared beam. Total activity: A needle mounted on a horizontal arm with a counterweight connected to the test cage registered all vibrations such as movements, grooming and shaking.
2. Materials and methods
All data were collected electronically through a computer interface. The animals were observed for a 60 min period of time (0–60 min) divided into three 20 min intervals; in each 60-min session animals from each exposure group were represented. A total of 12 males and 12 females per exposure group, where 3–4 individuals were taken randomly from at least 3 different litters, were observed for spontaneous behaviour.
2.1. Animals
2.4. Slot Blot analysis
Experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/ EEC), after approval from the local ethical committees (Uppsala University and the Agricultural Research Council) and by the Swedish Committee for Ethical Experiments on Laboratory Animals. Pregnant Naval Medical Research Institute (NMRI) mice were purchased from Scanbur, Sollentuna, Sweden. The animals were housed individually in plastic cages in a room with an ambient temperature of 22 8C and a 12/12 h constant light/dark cycle. Animals were supplied with standardized pellet food (Lactamin, Stockholm, Sweden) and tap water ad libitum. Females were checked for birth twice daily (08.00 and 18.00 h) and day of birth was designated day 0. Within the first 48 h after birth, litter sizes were adjusted to 10–12 pups of both sexes by euthanizing excess pups. At approximately 4 weeks of age, male and female offspring were separated with regard to sex and raised in sibling groups of 3–7 individuals in separate male and female rooms.
Male mice irradiated to a dose of 500 mGy were used in the Slot Blot analysis as they showed an altered spontaneous behaviour and lack of habituation. Sham irradiated mice were used as controls. The mice were sacrificed by cervical dislocation 24 h post-irradiation or at 6 months of age. The brains were dissected on an ice-cold glass plate. Cerebral cortex and hippocampus were collected, snap frozen in liquid nitrogen and stored at 80 8C until assayed. Both brain regions were homogenized in a RIPA cell lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 20 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1% sodium deoxycholate) to which a 0.5% protease inhibitor cocktail (Protease Inhibitor Cocktail set III, Calbiochem) was added. Homogenates were centrifuged at 14,000 g at 4 8C for 15 min and supernatant analyzed for protein content by using the BCA method (Pierce). Homogenates were stored at 80 8C. Viberg and co-workers have previously evaluated the specificity of antibodies CaMKII (Upstate Millpore, 05-552), GAP-43 (Chemicon Millipore, AB5220), synaptophysin (Calbiochem, 573822) and tau (Santa Cruz, 32274) by Western blot procedure with satisfactory results (Viberg et al., 2008; Viberg, 2009). The antibody used against tau recognizes both the nonphosphorylated and phosphorylated protein forms. Four mg of protein for CaMKII and GAP-43, 3 mg for synaptophysin and 3.5 mg for tau were diluted in sample buffer (120 mM KCl, 20 mM NaCl, 2 mM NaHCO3, 2 mM MgCl2, 5 mM HEPES, pH 7.4, 0.05% Tween20, 0.2% NaN3) to a final volume of 200 ml. Duplicates of each sample were applied to a nitrocellulose membrane (0.45 mm,
2.2. Irradiation Mice of both sexes were whole body gamma-irradiated on postnatal day 10 to a single dose from a 60Co source at The Svedberg laboratory, Uppsala University, Uppsala, Sweden (Eriksson et al., 2010). Mice were placed in plastic dishes and exposed to a single dose of 350 or 500 mGy with a surface dose rate of about 0.02 Gy/minute. An ionization chamber (Markus chamber type 23343, PTW-Freiburg) was used to measure the dose, which was
50
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
BioRad) using a Bio-Dot SF microfiltration apparatus (BioRad). Membranes were fixed in 25% isopropanol and 10% acetic acid solution for 15 min, washed and subsequently blocked for 1 h in 5% non-fat dry milk and 0.03% Tween-20 in room temperature. Incubation of membranes with primary mouse monoclonal CaMKII antibody (1:5000), rabbit polyclonal GAP-43 antibody (1:10,000), mouse monoclonal synaptophysin antibody (1:10,000) or mouse monoclonal tau antibody (1:1000) occurred overnight at 4 8C. A horseradish peroxidase conjugate secondary antibody against mouse (074-1806, 1:20,000) or rabbit (KPL 074-1506) was used to detect immunoreactivity. Immunoreactive bands were traced using an enhanced chemiluminescent substrate (Pierce, Super Signal West Dura) and imaged on a LAS-1000 (Fuji Film, Tokyo, Japan). Band intensity was quantified using IR-LAS 1000 Pro (Fuji Film). 2.5. Statistical analysis Data from the variables locomotion, rearing and total activity over the three consecutive 20-min time periods (treatment, time and treatment time, between subjects, within subjects and interaction factors, respectively) in the spontaneous behaviour in a novel home environment were submitted to a split-plot ANOVA design and pairwise testing using a Duncan’s MRT (multiple range test) test in SAS 9.1 software (Kirk, 1968; Festing, 2006; Eriksson, 2008; Lazic and Essioux, 2013). Evaluation of the Slot Blot analyses of CaMKII, GAP-43, synaptophysin and tau in cerebral cortex and hippocampus from male mice was made using one-way ANOVA, pairwise testing Duncan’s MRT (STATISTICA 10).
3. Results No overt visual signs of toxicity were observed in the irradiated mice throughout the experimental period. Nor were there any significant deviations in body weight between irradiated mice and control mice (data not shown). 3.1. Spontaneous behaviour in 2-month-old male mice The spontaneous behaviour variables locomotion, rearing and total activity in 2-month-old male mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 1. Significant treatment time interactions were observed (F4,66 = 220.47; F4,66 = 316.25; F4,66 = 144.45, p 0.001) for locomotion, rearing and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three test variables. Control males showed a normal decrease in activity over the 60 min period of testing time, as the novelty of the test chamber diminished, and habituated in a normal way, as earlier reported by Eriksson and co-workers (2010) and Fredriksson (1994). Male mice exposed to 350 mGy showed a significantly (p 0.01) decreased activity in the variables locomotion, rearing, and total activity during the first 20-min period, compared to control animals. Mice exposed to 500 mGy showed significantly (p 0.01) decreased locomotion, rearing and total activity during the first 20 min of testing, compared with control animals. Mice exposed to 500 mGy showed significantly (p 0.01) decreased locomotion and total activity during the first 20 min of testing, compared with animals exposed to 350 mGy. During the last 20 min of testing, mice exposed to 500 mGy showed significantly (p 0.01) increased locomotion, rearing, and total activity, compared to controls and animals exposed to 350 mGy (Fig. 1).
Fig. 1. Spontaneous behaviour of 2-month-old NMRI male mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 220.47; F4,66 = 316.25; F4,66 = 144.45) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (a) significantly different vs. control, p 0.05; (B) significantly different vs. 350 mGy, p 0.01. Height of bars represents mean SD.
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
51
3.2. Spontaneous behaviour in 2-month-old female mice The spontaneous behaviour variables locomotion, rearing, and total activity in 2-month-old female mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 2. Significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 134.87; F4,66 = 72.81, p 0.001) for locomotion, rearing, and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three-test variables. Control female mice showed normal decrease in activity and habituation capacity over the 60 min period of testing time, as seen in age-matched male mice. Females exposed to 350 mGy showed a significantly (p 0.01) decreased activity in the variables locomotion, rearing, and total activity during the first 20 min period, compared to control animals. Females exposed to 500 mGy showed significantly (p 0.01) decreased locomotion, rearing and total activity during the first 20 min of testing, compared with control females. Mice exposed to 500 mGy showed significantly (p 0.01) decreased locomotion and total activity during the first 20 min of testing, compared with females exposed to 350 mGy. During the last 20 min of testing, females exposed to 500 mGy showed significantly (p 0.01) increased locomotion, rearing, and total activity, compared to female controls and females exposed to 350 mGy (Fig. 2). 3.3. Spontaneous behaviour in 4-month-old male mice The spontaneous behaviour variables locomotion, rearing and total activity in 4-month-old male mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 3. Significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 1334.87; F4,66 = 72.8, p 0.01) for locomotion, rearing and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three test variables. Control males showed a normal decrease in activity and habituation capacity over the 60 min period of testing time, as was observed in 2-month-old male mice. Alterations in spontaneous behaviour observed at 4 months of age, following exposure to 350 mGy or 500 mGy, was in concordance with observed spontaneous behaviour alterations at 2 months of age and no additional significant changes were observed. 3.4. Spontaneous behaviour in 4-month-old female mice The spontaneous behaviour variables locomotion, rearing and total activity in 4-month-old female mice exposed to radiation (350 or 500 mGy) or sham irradiated (control) on PND 10 are presented in Fig. 4. Significant treatment time interactions were observed (F4,66 = 53.21; F4,66 = 93.68; F4,66 = 42.33, p 0.01) for locomotion, rearing and total activity, respectively. Pair-wise testing between the different doses of radiation and the sham irradiated control group showed significant dose-related changes in all three test variables. Control females showed a normal decrease in activity and habituation capacity over the 60 min period of testing time, as was observed in age-matched male mice and in 2-month-old females. Alterations in spontaneous behaviour observed in 4-month-old females, following exposure to 350 mGy or 500 mGy, was in concordance with observed spontaneous behaviour alterations in 2-month-old female mice and also age-matched males, with no additional significant changes being observed.
Fig. 2. Spontaneous behaviour of 2-month-old NMRI female mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 134.87; F4,66 = 72.81) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (B) significantly different vs. 350 mGy, p 0.01. Height of bars represents mean SD.
52
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
Fig. 3. Spontaneous behaviour of 4-month-old NMRI male mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 94.83; F4,66 = 1334.87; F4,66 = 72.81) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (a) significantly different vs. control, p 0.05; (B) significantly different vs. 350 mGy, p 0.01. Height of bars represents mean SD.
Fig. 4. Spontaneous behaviour of 4-month-old NMRI female mice irradiated with 350 mGy, 500 mGy or sham irradiated on PND 10. The data were subjected to an ANOVA with split-plot design and significant treatment time interactions were observed (F4,66 = 53.21; F4,66 = 93.68; F4,66 = 42.33) for locomotion, rearing, and total activity, respectively. Pairwise testing between control animals and animals exposed to radiation was performed using Duncan’s MRT test. The statistical differences are indicated as: (A) significantly different vs. control, p 0.01; (a) significantly different vs. control, p 0.05; (B) significantly different vs. 350 mGy, p 0.01; (b) significantly different vs. 350 mGy, p 0.05. Height of bars represents mean SD.
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
53
Table 1 Protein levels of CaMKII, GAP-43, synaptophysin and tau in male mice exposed to 500 mGy on postnatal day 10.a 11-day old
Control CaMKII GAP-43 Synaptophysin Tau
6-month-old
Cerebral cortex
Hippocampus
Cerebral cortex
Hippocampus
100 124 18 108 6 154 19** 218 14***
100 102 15 94 8 109 13 67 14**
100 114 24 100 10 114 10 205 17***
100 105 9 98 8 106 9 106 8
a Animals were sacrificed as neonatals 24 h post-irradiation or at the adult age of 6 months. Cerebral cortex and hippocampus were harvested and protein levels analysed using the Slot-Blot technique. The data were subjected to a one-way ANOVA, pairwise testing between control and 500 Gy exposed mice using Duncan’s test. The control value was set to 100% (SD) and the statistical difference between control and 500 Gy exposed mice is indicated with ** for p 0.01 or *** for p 0.001.
3.5. Analysis of levels of CaMKII, GAP-43 synaptophysin and tau in the cerebral cortex and hippocampus of 11-day-old and 6-month-old male mice At 11 days: Brain homogenates from hippocampus and cerebral cortex, collected 24 h post-irradiation, from male mice exposed on PND 10 to 0 or 500 mGy were analysed for levels of CaMKII, GAP-43, synaptophysin and total tau using the Slot-Blot technique. The results are presented in Table 1 as the percentage of control animal protein levels. Protein levels of sham irradiated animals were normalized to 100%. Significant (p 0.001) increase (118%) in tau levels as well as significant (p 0.01) increase (54%) in levels of synaptophysin were observed in cerebral cortex in male mice exposed to 500 mGy. Hippocampal levels of tau were significantly (p 0.01) decreased (33%) in male mice exposed to 500 mGy on PND 10, when compared to controls. At 6 months: Brain homogenates from cerebral cortex and hippocampus prepared from 6-month-old male mice, exposed on PND 10 to 0 or 500 mGy, were analysed for levels of the same neuroproteins as given above. The results are presented in Table 1 as the percentage of sham irradiated control animal protein levels, which were standardized to 100%. Six-month-old male mice, exposed to 500 mGy on PND 10, expressed significantly (p 0.001) increased (105%) levels of tau protein in cerebral cortex. No significant changes in neuroprotein levels were seen in the hippocampus (Table 1). 4. Discussion The present study shows that neonatal radiation exposure can cause similar persistent neurobehavioural deficits in male and female mice and that a threshold dose is seen around 350 mGy. Furthermore, in adult male mice showing reduced cognitive function, the level of total tau was increased in the cerebral cortex. An alteration in neuroprotein levels was also observed in neonatal mouse cerebral cortex and hippocampus following the whole body external irradiation. Male mice neonatally exposed to doses of 350 and 500 mGy displayed significantly aberrant spontaneous behaviour and a dose-response related decrease in habituation at the age of 2 and 4 months. During the spontaneous behaviour observation a decrease in sensoric stimuli, following the diminishment of the novelty of the test chamber rendered the control animals to respond less to these stimuli by displaying less activity and habituating to the novel home environment. Therefore, habituation can be used a measurement of cognitive function (Daenen et al., 2001; Rankin et al., 2009). Habituation, defined here as a decrease in the variables locomotion, rearing and total activity counts, was evident in control animals whereas mice irradiated with 500 mGy displayed reduced activity early in the 60 min test period, but a hyperactive behaviour toward the end. This
aberrant spontaneous behaviour in adult male mice following neonatal exposure to gamma radiation is in agreement with our earlier reported study (Eriksson et al., 2010). In that study a single dose of 500 mGy resulted in a loss of habituation to a novel home environment, accompanied by a hyperactive behaviour at the end of the observational period, while no effect was seen after a single dose of 200 mGy. In the present study a modified habituation was observed at 350 mGy, where a significant decrease in activity during the first 20-min period of testing indicates 350 mGy as a possible threshold value for induction of persistent neurobehavioural defects. Developmental exposure to radiation in studies by Hossain and Devi (2000, 2001) indicated persistent changes in adult mouse locomotor activity in the open-field test as well as memory and learning impairments following exposure to 500 mGy irradiation during the embryonic period of brain development (Hossain and Devi, 2000, 2001). Multiple studies conducted to investigate neurotoxic effects following internal exposure to uranium in adult animals have shown that exposure to depleted uranium causes alterations in sleep-wake cycles as well as food intake, while exposure to enriched uranium resulted in additional alterations in sleeping patterns as well as impairments in spatial working memory and higher levels of anxiety-like behaviour in rats (Houpert et al., 2005; Lestaevel et al., 2005a, 2005b). Houpert and co-workers (2005) suggest that the radiation from enriched uranium is the main inducer of the observed disruptions in working memory and anxiety-like behaviour since exposure to depleted uranium for an equivalent time period did not result in similar behavioural alterations. In female mice the spontaneous behaviour test also revealed a dose-response related disruption of habituation capacity in both 2-month-old and 4-month-old mice following neonatal irradiation to 350 or 500 mGy. This effect was similar to the altered habituation observed in the age-matched male mice. This shows that female mice can be as susceptible as male mice to develop aberrant spontaneous behaviour and loss of habituation to a novel home environment following a single neonatal exposure to radiation. Furthermore, this indicates that there are no major differences between sexes regarding manifestation of this type of developmental neurotoxic defects in mice exposed to radiation. Other studies suggest that female mice, acutely irradiated with 8 Gy while under anaesthesia on PND 14, develop more pronounced manifestations of impaired white matter growth, impaired hippocampal neurogenesis and anxiety at an adult age when compared to males (Roughton et al., 2012, 2013). One cannot rule out a possible interaction effect between radiation and the anaesthetic as a causative factor for the sex differences observed by Roughton and co-workers. Whether there are sex differences in mechanisms of induction of neurotoxicity of high dose exposure compared to low dose exposure to radiation remains to be investigated.
54
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55
During the BGS a distinct ontogeny of several important neuroproteins such as CaMKII, GAP-43, synaptophysin and tau can be observed, where the most pronounced increase in proteins occurs around PND 7–14 (Viberg et al., 2008; Viberg, 2009). The present study show elevated levels of synaptophysin and tau in cerebral cortex of neonatal male mice exposed to a single dose of 500 mGy, but also a decrease in hippocampal total tau levels. Changes in levels of neuroproteins have been reported following adult exposure to radiation. A reduction in CaMKII levels in adult male mice, but not female was observed, following 500 mGy X-ray irradiation at the adult age of 45 day (Silasi et al., 2004). Interestingly, Goutan and co-workers (1999) found a decrease in cerebellar GAP-43 levels but no alterations in synaptophysin were observed following an acute 2 Gy whole body gamma radiation dose on PND 1. This alteration was not present 48 h postirradiation indicating a capability of the cerebellum to recover after irradiation during postnatal development (Goutan et al., 1999). Keeping in mind the distinct ontogeny of CaMKII, GAP-43, synaptophysin and tau during the BGS, it is reasonable to assume that disruptions in protein levels and the ability of the brain to recover from insults may vary depending at which time-point of brain development the exposure occurs (Viberg et al., 2008; Viberg, 2009). A defined critical period, around PND 10 in mouse, for induction of persistent behavioural defects, including altered cognitive function and changes in cholinergic receptors, has been seen after exposure to different types of chemicals (Eriksson et al., 1992, 2000; Eriksson, 1997). It is worth noting, that the elevated level of tau protein observed in cerebral cortex of neonatal male mice is still present in 6-month-old animals. Alzheimer’s disease (AD) is characterized by a progressive and profound loss of cognitive functions presumably attributed to cholinergic system dysfunction in early stages and the presence of intracellular neurofibrillary tangles consisting of hyper-phosphorylated aggregates of tau proteins in later stages of disease progression (Francis et al., 1999; Hardy and Selkoe, 2002; Di Domenico et al., 2011). An elevated level of phosphorylated tau protein in cerebrospinal fluid is used in medicine as a diagnostic marker for AD (Ikeda et al., 2013). However, in this study alterations in neuroproteins and behaviour were prominently present at higher radiation doses (500 mGy) than received during a single CT scan, which has been estimated to deliver an average dose of 30 mGy to the brain (Pearce et al., 2012b). Still it is important to consider possible late neurocognitive defects deriving from irradiation of non-target tissue in radiotherapy of brain tumours in childhood (Mulhern et al., 2004), as well as repeated CT scans in children. It has been estimated that approximately 30% of patients younger than 22 years old underwent more than one CT scan in Great Britain during the years 1993–2002 (Pearce et al., 2012a). Taking into consideration that the radiation dose delivered to the brain during a single CT scan can vary from 21 to 153 mGy, according to a recent Swedish report form the Radiation Safety Authority (Leitz and Alme´n, 2010), the tentative threshold dose (350 mGy) observed to induce spontaneous behaviour alterations in this study is well within the range of a plausible exposure situation. 5. Conclusion The present study shows that irradiation to a single dose of 500 mGy can cause developmental neurotoxic effects, similar in both male and female mice, manifested as a lack of or reduced capacity to habituate to a novel home environment. Moreover, irradiation to a dose of 350 mGy seems to be a tentative threshold for induction of this type of neurotoxicity. Neonatal exposure to 500 mGy caused increased levels of tau protein in the cerebral cortex of neonatal male mice. This trait was also observable in
adult male mice, showing reduced cognitive function. The finding of increased levels of total tau protein in mice calls for further investigation, as phosphorylated tau is a diagnostic marker in humans with AD. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This work was supported by the European Community’s Seventh Framework Programme (EURATOM) [CEREBRAD; grant number 29552] and the Swedish Radiation Safety Authority. References Abreu-Villaca Y, Filgueiras CC, Manhaes AC. Developmental aspects of the cholinergic system. Behav Brain Res 2011;221:367–78. Benowitz LI, Routtenberg A. GAP-43: an intrinsic determinant of neuronal development and plasticity. Trends Neurosci 1997;20:84–91. Bernier MO, Rehel JL, Brisse HJ, Wu-Zhou X, Caer-Lorho S, Jacob S, et al. Radiation exposure from CT in early childhood: a French large-scale multicentre study. Br J Radiol 2012;85:53–60. Bolles RC, Woods PJ. The ontogeny of behaviour in the albino rat. Anim Behav 1964;12: 427–41. Campbell BA, Lytle LD, Fibiger HC. Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science 1969;166:635–7. Daenen EWPM, Van der Heyden JA, Kruse CG, Wolterink G, Van Ree JM. Adaptation and habituation to an open field and responses to various stressful events in animals with neonatal lesions in the amygdala or ventral hippocampus. Brain Res 2001;918: 153–65. Davison AN, Dobbing J. Applied neurochemistry. Oxford: Blackwell; 1968. Di Domenico F, Sultana R, Barone E, Perluigi M, Cini C, Mancuso C, et al. Quantitative proteomics analysis of phosphorylated proteins in the hippocampus of Alzheimer’s disease subjects. J Proteomics 2011;74:1091–103. Eriksson P. Developmental neurotoxicity of environmental agents in the neonate. Neurotoxicology 1997;18:719–26. Eriksson P. Response to: use of the pup as the statistical unit in developmental neurotoxicity studies: overlooked model or poor research design? Toxicol Sci 2008;103:411–3. Eriksson P, Ahlbom J, Fredriksson A. Exposure to DDT during a defined period in neonatal life induces permanent changes in brain muscarinic receptors and behaviour in adult mice. Brain Res 1992;582:277–81. Eriksson P, Ankarberg E, Fredriksson A. Exposure to nicotine during a defined period in neonatal life induces permanent changes in brain nicotinic receptors and in behaviour of adult mice. Brain Res 2000;853:41–8. Eriksson P, Fischer C, Stenerlow B, Fredriksson A, Sundell-Bergman S. Interaction of gamma-radiation and methyl mercury during a critical phase of neonatal brain development in mice exacerbates developmental neurobehavioural effects. Neurotoxicology 2010;31:223–9. Erondu NE, Kennedy MB. Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain. J Neurosci 1985;5:3270–7. Festing MFW. Design and statistical methods in studies using animal models of development. Ilar J 2006;47:5–14. Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic hypothesis of Alzheimer’s disease: a review of progress. J Neurol Neurosurg Psychiatry 1999;66:137–47. Fredriksson A. MPTP-induced behavioural deficits in mice: validity and utility of a model of parkinsonism. Uppsala: Uppsala University; 1994. Goutan E, Marti E, Ferrer I. Expression of synaptic proteins in the developing rat cerebellum following ionizing radiation. Int J Dev Neurosci 1999;17:275–83. Hall P, Adami HO, Trichopoulos D, Pedersen NL, Lagiou P, Ekbom A, et al. Effect of low doses of ionising radiation in infancy on cognitive function in adulthood: Swedish population based cohort study. Br Med J 2004;328:19. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 2002;297:353–6. Hossain M, Devi PU. Effect of irradiation at the early fetal stage on adult brain function in the mouse: locomotor activity. Int J Radiat Biol 2000;76:1397–402. Hossain M, Devi PU. Effect of irradiation at the early foetal stage on adult brain function of mouse: learning and memory. Int J Radiat Biol 2001;77:581–5. Houpert P, Lestaevel P, Bussy C, Paquet F, Gourmelon P. Enriched but not depleted uranium affects central nervous system in long-term exposed rat. Neurotoxicology 2005;26:1015–20. Ikeda M, Yonemura K, Kakuda S, Tashiro Y, Fujita Y, Takai E, et al. Cerebrospinal fluid levels of phosphorylated tau and Abeta1–38/Abeta1–40/Abeta1–42 in Alzheimer’s disease with PS1 mutations. Amyloid 2013;20:107–12. Kirk RE. Procedures for the behavioural science. Belmont, CA: Brooks/Cole; 1968. Lazic SE, Essioux L. Improving basic and translational science by accounting for litterto-litter variation in animal models. BMC Neurosci 2013;14:37.
S. Buratovic et al. / NeuroToxicology 45 (2014) 48–55 Leitz W, Alme´n A. Patientdoser fra˚n ro¨ntgenunderso¨kningar i Sverige – utveckling fra˚n 2005 till 2008. Swedish Radiation Safety Authority; 2010. Lestaevel P, Bussy C, Paquet F, Dhieux B, Clarencon D, Houpert P, et al. Changes in sleepwake cycle after chronic exposure to uranium in rats. Neurotoxicol Teratol 2005a;27:835–40. Lestaevel P, Houpert P, Bussy C, Dhieux B, Gourmelon P, Paquet F. The brain is a target organ after acute exposure to depleted uranium. Toxicology 2005b;212:219–26. Lisman J, Schulman H, Cline H. The molecular basis of CaMKII function in synaptic and behavioural memory. Nat Rev 2002;3:175–90. Mettler FA Jr, Wiest PW, Locken JA, Kelsey CA. CT scanning: patterns of use and dose. J Radiol Prot 2000;20:353–9. Mulhern RK, Merchant TE, Gajjar A, Reddick WE, Kun LE. Late neurocognitive sequelae in survivors of brain tumours in childhood. Lancet Oncol 2004;5:399–408. Oestreicher AB, De Graan PN, Gispen WH, Verhaagen J, Schrama LH. B-50, the growth associated protein-43: modulation of cell morphology and communication in the nervous system. Prog Neurobiol 1997;53:627–86. Pearce MS, Salotti JA, Howe NL, McHugh K, Kim KP, Lee C, et al. CT scans in young people in Great Britain: temporal and descriptive patterns, 1993–2002. Radiol Res Pract 2012;594278. Pearce MS, Salotti JA, Little MP, McHugh K, Lee C, Kim KP, et al. Radiation exposure from CT scans in childhood and subsequent risk of leukaemia and brain tumours: a retrospective cohort study. Lancet 2012b;380:499–505. Rankin CH, Abrams T, Barry RJ, Bhatnagar S, Clayton DF, Colombo J, et al. Habituation revisited: an updated and revised description of the behavioral characteristics of habituation. Neurobiol Learn Mem 2009;92:135–8.
55
Roughton K, Bostrom M, Kalm M, Blomgren K. Irradiation to the young mouse brain impaired white matter growth more in females than in males. Cell Death Dis 2013;4:e897. Roughton K, Kalm M, Blomgren K. Sex-dependent differences in behavior and hippocampal neurogenesis after irradiation to the young mouse brain. Eur J Neurosci 2012;36:2763–72. Sarnat HB, Born DE. Synaptophysin immunocytochemistry with thermal intensification: a marker of terminal axonal maturation in the human fetal nervous system. Brain Dev 1999;21:41–50. Silasi G, Diaz-Heijtz R, Besplug J, Rodriguez-Juarez R, Titov V, Kolb B, et al. Selective brain responses to acute and chronic low-dose X-ray irradiation in males and females. Biochem Biophys Res Commun 2004;325:1223–35. Wang JZ, Liu F. Microtubule-associated protein tau in development, degeneration and protection of neurons. Prog Neurobiol 2008;85:148–75. Viberg H. Neonatal ontogeny and neurotoxic effect of decabrominated diphenyl ether (PBDE 209) on levels of synaptophysin and tau. Int J Dev Neurosci 2009;27: 423–9. Viberg H, Mundy W, Eriksson P. Neonatal exposure to decabrominated diphenyl ether (PBDE 209) results in changes in BDNF, CaMKII and GAP-43, biochemical substrates of neuronal survival, growth, and synaptogenesis. Neurotoxicology 2008;29: 152–9. Yamauchi T. Neuronal Ca2+/calmodulin-dependent protein kinase II – discovery, progress in a quarter of a century, and perspective: implication for learning and memory. Biol Pharm Bull 2005;28:1342–54.