{'fraction_non_alphanumeric': 0.03714073076514158, 'fraction_numerical': 0.02991104653223234, 'mean_word_length': 4.714459295261239, 'pattern_counts': {'":': 0, '<': 17, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 1, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
X-irradiation of developing hippocampal neurons causes changes in neuron population phenotypes, dendritic morphology and synaptic protein expression in surviving neurons at maturity

The effects of X-irradiation on developing neurons and their functions are unclear. We used primary cultures of mouse hippocampal neurons to investigate the effects of X-irradiation on cell death in developing neurons by analyzing caspase-3, MAP2 and DAPI-labeled cells, and the phenotypes and function of surviving neurons, by examining GAD67-positive cells as a GABAergic marker, and the synaptic markers synapsin 1, drebrin and PSD-95 through its maturation. One-day in vitro (DIV 1) cells were exposed to 0.5 Gy or 1 Gy of X-rays. A significant increase in the percentage of activated caspase-3, a decrease in the number of MAP2/DAPI-positive cells and change in the percentage of GAD67 positive neurons, compared with sham controls, were found 6 days after 1 Gy and 13 days after 0.5 Gy of X-rays. The expression of PSD-95 and drebrin, as well as drebrin clusters, in the remaining neurons was decreased at DIV 21, in both 0.5 Gy and on 1 Gy-irradiation there was a reduced number of dendritic intersection as well. Together, our findings show that 0.5 Gy and 1 Gy of X-irradiation at DIV 1 not only causes neuronal cell death but elicits an increase in the percentage of inhibitory neurons, changes in the dendrites and decrease in expression of important synaptic proteins in the surviving neurons at maturity 3 weeks after exposure.


Introduction
The effects of radiation on the nervous system are of major interest because of the widespread use of radiation in medicine for diagnostic and therapeutic purposes in patients of various ages and neurological status. Mature neurons are non-dividing cells, and are therefore considered relatively radio-resistant compared with actively dividing cells. Several studies on the effects of radiation on cells, including immature neurons, have focused on radiationinduced acute cell death, i.e., within 48 h, without examining the long-term effects on the surviving neurons (Ferrer, 1999;Michelin et al., 2004;Shirai et al., 2006;Kaminuma et al., 2010). Therefore, the effects on mature neurons of early radiation exposure of imma-ture neurons are unclear. Immature neurons are found not only in the developing brain, but also in certain regions of the adult brain. For example, in the adult brain, adult neurogenesis continually produces new neurons in the hippocampus. Following neurogenesis, the integration of the new neurons into the existing neural circuitry is critical for cognitive function (van Praag et al., 1999). Recently, adult neurogenesis has been found to have an important role in functional cognitive recovery after traumatic brain injury (Perederiy et al., 2013;Villasana et al., 2015). Moreover, in the developing and adult brain, the immature neurons undergo synaptic changes as they mature to produce functionally mature synapses.
Although mature neurons in the brain are considered resistant to radiation-induced cell death, it has been reported that radiation alters the numbers of some types of dendritic spines (Parihar and Limoli, 2013;Shirai et al., 2013) and induces changes in postsynaptic protein clusters along the dendrite that may lead to synaptic dysfunction or synaptopathy (Okamoto et al., 2009;  2013). Because radiation might also affect synaptic function, studies are needed to elucidate the long-term impact of radiation on immature neurons by not only examining cell death but also synaptic proteins in surviving neurons. In the present study, we exposed DIV 1 primary mouse hippocampal neuronal cultures to 0.5 Gy or 1 Gy of X-rays, and examined the effects on cell death and synaptic protein expression at DIV 4, 7, 14 and 21 after irradiation.

Mouse primary hippocampal cultures
All animal experiments were performed according to guidelines set by the Animal Care and Experimentation Committee of Gunma University, Showa Campus, Maebashi, Japan. Every effort was made to minimize animal suffering and minimize the number of animals used.

Experimental design
On DIV 1, the cultured cells were irradiated with a single dose of 0.5 Gy or 1 Gy X-rays. We confirmed the cells were still immature by immunolabeling for doublecortin (DCX), a marker of immature neurons (Fig. 1a). At the time (DIV 1) of irradiation, 90 % of the cells were neurons, according to MAP2 immunostaining and DAPI labeling. MAP2-negative/DAPI-labeled cells were regarded as nonneuronal cells (Fig. 1b). The cells were cultured for another 1, 3, 6, 13 or 20 days (Fig. 1c). The percentage of activated caspase-3-positive cells was calculated at these time points. Analysis of synaptic proteins was performed on DIV 14 and 21, when the neurons are undergoing synaptogenesis (Goslin and Banker, 1989;Papa et al., 1995).
Next, the impact of irradiation on synaptic protein expression was examined. Drebrin and PSD-95 were used as postsynaptic markers, while synapsin I was used as a presynaptic marker in mature neurons, i.e., DIV 14 and 21. Protein levels were quantified by western blotting, and the accumulation of drebrin along the dendrites was assayed by immunocytochemistry. The cells irradiated on DIV 1 were fixed on DIV 14 (dendritic spine formation phase) and 21 (dendritic spine maintenance phase). All experiments were performed in triplicate.

X-irradiation
X-rays were generated using a Shimadzu X-TITAN 225S Xray generator (Shimadzu Inc., Kyoto, Japan), and a dose rate of 1.3 Gy/min was used to irradiate the cells. The distance between cells and the radiation source was approximately 473 mm, and the irradiated samples received a single absorbed dose of 0.5 Gy or 1 Gy. Sham-irradiated samples were transported to the radiation facility but not exposed to radiation.

Immunocytochemistry
For each experiment, one or two coverslips containing cells were used for each time point, at day 1 (DIV1), the day of irradiation, and 3 days (DIV 4), 6 days (DIV 7), 13 days (DIV 14) and 20 days (DIV 21) following the irradiation. All cultures were from the original 12well plate. Cells were fixed with 4 % paraformaldehyde in phosphate buffer (PB), pH 7.4, at 4 • C for 20 min. After permeabilization with 0.1 % Triton X-100, the cells were blocked with 3 % bovine serum albumin in PBS for 60 min, and then incubated overnight at 4 • C with primary antibodies. After washing, the cells were incubated for 1 h with the secondary antibodies, rinsed with PBS, and then mounted.

Immunofluorescence microscopy
Images were obtained with a conventional fluorescence Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) using MetaMorph microscopy automation and image analysis software (Meta Imaging V7.7; RRID: SciRes 000136; Molecular Devices, Sunnyvale, CA). Images were acquired at an excitation wavelength of 568 nm for MAP2 and 488 nm for activated caspase-3, and with an ultraviolet laser for DAPI. Each region of interest (ROI) was selected randomly and observed at 341 nm/pixel (1024 × 768 pixels) with a 10× objective (numerical aperture, 0.70) for the surviving neuron assay and with a 20× objective (numerical aperture, 0.70) for the quantification of active caspase-3-positive cells. Images for drebrin cluster quantification were obtained at 341 nm/pixel (1024 × 768 pixels) through a 63× objective (numerical aperture, 0.70). All images were downloaded to a computer using MetaMorph v7.10. After calibration of each image, the number of cells and drebrin puncta along the dendrite were calculated automatically.

Sholl analysis
The neurons were traced, and the Sholl analysis was obtained with an open-source program for ImageJ/Fiji2 (Schindelin et al., 2012;Ferreira et al., 2014) calculating the cumulative number of dendritic intersections at ten m interval distance points starting from the cell body (SHOLL, 1953). A total 18 neurons, 23 neurons, and 23 neurons for each sham, 0.5 Gy, and 1 Gy samples were analyzed, respectively.

Quantification of the number of neurons and active caspase-3 positive cells
For quantification of the number of immature neurons, the cultured neurons were fixed at DIV 1 and immunolabeled with DCX (a marker of immature neurons) or MAP2 to assess the number of immature neurons before irradiation ( Fig. 1a and b). The cultured neurons were irradiated with a single 0.5 or 1 Gy dose of X-rays at DIV 1. Then, cells were fixed at DIV 4 (i.e., 3 days after irradiation) before cytarabine treatment to exclude a potential impact of this drug on the number of irradiated cells, DIV 7 (6 days after irradiation), DIV 14 (13 days after irradiation), and DIV 21 (20 days after irradiation), as shown in the experimental design (Fig. 1c). We immunolabeled for the neuronal marker MAP2 to identify neurons. Images were obtained from 15 random ROIs. To assess apoptosis, the cultured neurons were fixed at DIV 2, 7 and 14, and the percentage of caspase-3-positive/DAPI-labeled cells among the total number of DAPI-labeled cells was calculated (more than 300 cells for each experiment).

Analysis of drebrin clusters
Drebrin clusters were defined as an immunolabeled region with a peak fluorescence intensity 2-fold higher than the average fluo-rescence intensity of dendrites, as previously described (Takahashi et al., 2009). These puncta, representing drebrin clusters, were counted along a 50-55-m length of a randomly selected dendrite 10 m distal to the cell body. Total 40 neurons were analyzed per dose per experiment.

Western blot analysis
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were performed as described previously. Briefly, cells from 12 coverslips were harvested in 240 l cell lysis buffer (10 mM Tris−HCl, 150 mM NaCl, 2 % SDS, 20 mM NaF, 1 mM Na 3 VO 4 ). After solubilization, the protein concentrations were determined using the DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (2.5-5 g/lane) were subjected to SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride membranes (Merck Millipore). The membranes were incubated with the appropriate primary and secondary antibodies. Peroxidase activity was detected using chemiluminescence reagents (Immobilon Western Chemiluminescent HRP Substrate, Merck Millipore) and visualized with an image analyzer (LAS-3000, Fujifilm, Tokyo, Japan). The protein levels were quantified with NIH Image J software after stan-dardizing the ratio of drebrin/total GAPDH, PSD95/ total GAPDH, and Synapsin I/ total H3.

Statistical analysis
Analyses were performed with GraphPad Prism 8. Unpaired two-tailed Student's t-tests relative to sham or comparisons between more than two groups were performed using analysis of variance followed by Bonferroni tests to determine significance. Statistical significance was assigned *p < 0.05, **p<0.01.

Irradiation of developing neurons causes a delayed decrease in the number of neurons
About 90 % of the cells were MAP2-positive (i.e., neurons) on DIV 1 and were immature ( Fig. 1a and b) at the time of irradiation. We analyzed the number of neurons at DIV 4 (3 days after irradiation) and found no significant difference in the number of cells for either dose, compared with sham cultures ( Fig. 2a and b). However, the number of neurons was significantly decreased in cultures exposed to 1 Gy of X-rays at DIV 7, 14 and 21, compared with sham cultures (P < 0.01; Fig. 2a and b). Cultures that received 0.5 Gy showed significant decreases at DIV 14 and 21, compared with sham cultures (P < 0.01; Fig. 2a and b). At DIV 21, although both doses produced significant decreases in the number of neurons, compared with sham cultures, the numbers of neurons in the 0.5 Gy and 1 Gy-irradiated cultures were similar to those at DIV 14 as were the numbers in sham cultures (Fig. 2b). This result indicates that the number of neurons is relatively constant from DIV 14-21. Most cells at DIV 1 (24 h after seeding) are morphologically in stages 2 or 3, which are cells with an outgrowth of minor processes (Dotti et al., 1988;Ohara et al., 2015). At these stages, neurons are still actively extending neurites and are not yet mature. In vivo studies in rats show that the rapid decline in the number of neurons at postnatal day 12 helps optimize neural function (Janec and Burke, 1993;Jackson-Lewis et al., 2000), in line with our current in vitro observation that in sham-treated cultures, the number of neurons is reduced at two weeks.

Activated caspase-3 levels increase concurrently with delayed cell death
Previous reports show that caspase-3 activity increases with radiation dose and is maximal at 4-6 h after 3 or 4 Gy of ␥irradiation in DIV 5 neural precursor cells from developing rat brain (Michelin et al., 2004). Therefore, we examined whether there is an increase in caspase-3 activity after X-irradiation of immature neurons in vitro. Although there was no significant difference in the percentage of caspase-3-positive cells at DIV 2 in any group, the percentage of caspase-3-positive cells was increased in the 1 Gy Xirradiated cultures at DIV 7, and in 0.5 Gy X-irradiated cultures at DIV 14, compared with sham (P < 0.01; Fig. 3a and b). However, the percentage of active caspase-3-positive cells was not significantly different between sham and 0.5 Gy X-irradiated cultures at DIV 7 or between sham and 1 Gy X-irradiated cultures at DIV 14 ( Fig. 3a  and b). These results suggest that X-irradiation induces a delayed increase in active caspase-3 levels in a dose and time-dependent manner.

Change in the population of inhibitory neurons among remaining irradiated neurons
In the hippocampus, GABAergic inhibitory interneurons represent only ∼10-15 % and the rest of the neuron population is glutamatergic excitatory neurons (Woodson et al., 1989;Aika et al., 1994). To distinguish the types of remaining neurons after irradiation, we determined the number of inhibitory neurons using GAD67-immunoreactivity as the marker of inhibitory neurons (Fig. 4a). The number of GAD67 positive neuron were analyzed at DIV 1, the time of irradiation, one week and two weeks after irradiation, and then we calculated the percentage of inhibitory neurons in the total population. There were no differences in the total number or percentage of GAD67 positive neurons at DIV 1, the day of irradiation (Fig. 4b). After one week of incubation, the number of GAD67 positive neurons was significantly decreased by 0.5 Gy and 1 Gy of irradiation compared to the sham, and a decrease in the fraction of inhibitory neurons was found after 0.5 Gy while an increase in the fraction of inhibitory neurons was found after 1 Gy (*P < 0.05; **P < 0.01; Fig. 4c). After two weeks of incubation, the total number and fraction of remaining GAD67 positive neurons were increased significantly after 0.5 Gy and 1 Gy of X-irradiation (**P < 0.01; Fig. 4a and d).

X-irradiation of immature neurons causes changes in dendrite arborization and synaptic proteins
Dendritic morphology is one of the vital factors for neuropathological condition (Yamada et al., 1988;Moolman et al., 2004) and disorders associated with mental retardation (Dierssen and Ramakers, 2006). Therefore, we analyzed the dendritic arborization when neurons reach maturation at DIV 21. We found that 1 Gy of irradiation significantly altered the number of intersections (P < 0.01; Fig. 5a-c).
The actin-binding protein drebrin is considered a regulator of dendritic spine morphogenesis and synaptic activities. Several studies have shown that drebrin plays pivotal roles in spine formation and synaptic plasticity (Aoki et al., 2005(Aoki et al., , 2009Jung et al., 2015). Drebrin accumulates in the dendritic spines when the filopodia form (Takahashi et al., 2009). Synaptic plasticity also requires numerous other proteins, including the important scaffolding protein PSD-95. PSD-95 is localized to dense specialized regions within the dendritic spines of excitatory synapses and is associated with synaptic plasticity (El-Husseini et al., 2000). Therefore, we analyzed synaptic proteins when the filopodia start to develop, at DIV 14, and when they are mature, at DIV 21. Western blot analysis showed that there was no change in the levels of drebrin, PSD-95 or synapsin 1 examined at DIV 14 ( Fig. 6a-c). However, surprisingly, at DIV 21, both doses of radiation produced significant decreases in drebrin compared with the sham cultures (P < 0.05, P < 0.01; Fig. 6d). Although 0.5 Gy had a tendency to decrease PSD-95 levels, the change was not significant. In comparison, significant reductions in PSD-95 were found in neurons exposed to 1 Gy X-rays at DIV 21 (P < 0.01; Fig. 6e). For functional synapses, the presynaptic nerve terminal also needs to be properly functioning. We therefore examined the presynaptic protein synapsin 1. Synapsin 1 is present in the majority of nerve terminals, where it is primarily associated with synaptic vesicles (De Camilli et al., 1983;Goslin and Banker, 1989). Interestingly, there was no change in synapsin 1 levels at DIV 14 or 21 in any treatment group (Fig. 6c and f).
Next, we analyzed the number of drebrin clusters along the dendrites of DIV 14 and DIV 21 neurons by immunocytochemistry. The number of drebrin clusters was not different at DIV 14 (13 days following irradiation) ( Fig. 7a and b) for either dose. However, the number of drebrin clusters was decreased at DIV 21 in neurons exposed to 1 Gy of radiation, but not in those exposed to 0.5 Gy (P < 0.01; Fig. 8a and b). These results indicate that X-irradiation affects postsynaptic proteins, but not presynaptic proteins, in mature neurons 20 days after radiation exposure while the neurons were immature.

Discussion
The main findings of the present study are the following: (1) Exposure of immature (DIV 1) primary mouse hippocampal neu-rons to 0.5−1 Gy of X-rays does not acutely alter the number of neurons, but does cause a gradual reduction in the number of neurons in tandem with an increase in the percentage of activated caspase-3-positive cells; (2) The X-irradiation changes the percentage of inhibitory neurons in the population of remaining neurons; and (3) The X-irradiation not only changes the number of intersections of remaining neurons but also the expression levels of the postsynaptic proteins drebrin and PSD-95 are reduced significantly by 20 days following irradiation, but are not reduced during the immature (spine formation) stage 13 days following irradiation. The western blotting results are supported by immunocytochemical analysis of drebrin clusters along the dendrite at 20 days following exposure to 1 Gy of X-rays. It is noteworthy that the reductions in both drebrin and PSD-95 occur 20 days following exposure to 1 Gy of radiation although there are no changes in cell number from DIV14 to DIV 21.
The developing brain is considerably more vulnerable to the effects of radiation than the adult brain. In the hippocampus, newly generated neurons have essential roles in cognitive function. While clinicians try to avoid irradiating the hippocampus, this is difficult in actual practice. We show here that radiation causes delayed decreases in the number of remaining neurons, which may result in a reduction in hippocampal volume (Lv et al., 2018). Previous studies have shown that 2 Gy of X-irradiation of the whole brain young adult mice causes an acute decrease in the number of Ki-67-positive proliferating cells and in the number of DCX-positive immature neurons (Mizumatsu et al., 2003;Raber et al., 2004). Other reports show that brain X-irradiation does not cause DCX-positive cells to  The Sholl analysis of the number of intersections and total of intersections within 120-m from the soma, respectively. There was a decrease in the number of intersections after 1 Gy of irradiation. The difference in the number of intersections between sham and treatment in each irradiated group was analyzed using multiple comparison correction. **P < 0.01. Each point represents the mean ± SEM for 18-23 neurons from three independent experiments. completely disappear within 8 and 24 h of exposure (Puspitasari et al., 2016;Miao et al., 2018), and that some neurons remain viable 7 and 14 days after irradiation at DIV 7 in vitro (Okamoto et al., 2009). At least 2 Gy of ␥-irradiation is required to cause the death of very young (DIV 0.5) neurons within 24 h. In more mature DIV 7 neurons, at least 4 Gy of X rays is needed to increase apoptosis and decrease drebrin clusters along dendrites 2 weeks after irradiation, although it does not affect the number of neurons (Okamoto et al., 2009). This suggests that the sensitivity of neurons to radiation is dependent on the dose and developmental period. Therefore, to determine the fate of immature neurons without killing the neurons we analyse following less than 2 Gy of X-rays, we used 0.5 Gy and 1 Gy of X-rays.

X-irradiation alters the number of neurons and percentage of GAD 67 positive immature neurons
During normal development, neurons undergo cell death (Pfisterer and Khodosevich, 2017). Indeed, we found here that nonirradiated (sham-treated) neurons also decrease in number over time. The 0.5 and 1 Gy doses of X-irradiation enhanced cell death, leading to a significant reduction in the number of neurons within 13 days after irradiation, compared with sham treatment. While neurons that received 0.5 Gy decreased in number within 13 days, those that received 1 Gy decreased in number within 6 days, in tandem with the increase in the percentage of activated caspase-3-positive cells. These results indicate that doses as low as 0.5 and 1 Gy increase cell death in developing immature neurons in a time and dose-dependent manner. Apoptosis can occur by a variety of biochemically distinct mechanisms, and the timeline of apoptotic events depends on various factors, including the type of cells (Chen and Sakai, 2004;Wichmann et al., 2006). In this study, we showed that the activation of caspase-3 in irradiated immature neurons occurs later than 24 h. Apoptosis of neurons is a normal event needed for the optimisation of neural function (Dekkers et al., 2013). In this study, the radiation-induced caspase-3 activation 1-2 weeks after exposure may indicate that neurons attempt to survive in the first few days, but are eventually unable to do so. A previous study using low doses of ␥-rays on DIV 0.5 primary neurons, when the cells are in stage 1-2, showed a decrease in some cells exposed to 2 Gy within 24 h, compared with sham-irradiated controls (Yang et al., 2011). Another study compared the radiosensitivity of devel- The difference in the fold-change between sham and treatment in each irradiated group was analyzed using Student's t-test with Bonferroni correction. *P < 0.05, **P < 0.01. Each point represents the mean ± SEM for three different fields from three independent experiment. oping (DIV 7) and mature (DIV 21) neurons 24 h following 30 Gy of X-irradiation, and showed that immature neurons were sensitive to the radiation, while mature neurons were resistant, with no significant apoptosis (Shirai et al., 2006).
Neuronal disorders such as epilepsy, autism and schizophrenia are often explained as being caused by an imbalance of the excitatory and inhibitory circuits in the brain (Gao and Penzes, 2015;Selten et al., 2018). In normal conditions, hippocampus GABAergic local circuit inhibitory interneurons represent only ∼10-15 % of the total neuronal population (Woodson et al., 1989;Aika et al., 1994). In this study, at DIV one the number and percentage of GAD67 positive neurons are found to be low, but they increase after one week of incubation including in the irradiated cells. However, both radiation doses result in a smaller number of GAD67 positive cells, compared to the un-irradiated control, and alter the percentage of remaining GAD67 neurons. At one week the percentage of GAD67-positive neurons after 0.5 Gy was found to be decreased because the total number of neurons was not yet decreased by the irradiation. On the other hand, with 1 Gy irradiated neurons the percentage of GAD67-positive neurons was slightly increased due to the decrease in the total number of neurons. After two weeks, DIV 14, the number of GAD67-positive neurons was decreased in the sham compared to the one week old neurons, but the number and percentage was increased after 0.5 Gy and 1 Gy of irradiation. These data suggest that irradiation significantly alters the percentage of the GABAergic neurons in the total remaining neuronal population within two weeks after 1 Gy irradiation up to almost 30 per cent. It is noteworthy that after radiation some GABAergic neurons remain after two weeks even though their number was decreased in the sham treated cells. In a previous study using 4month old tgCRND8 and 18-month old APP/PS1 transgenic mice, respectively, both glutamatergic and GABAergic presynaptic terminals were found elevated at an early stage in the distinct Alzheimer Disease (AD) mice models (Bell et al., 2003(Bell et al., , 2006. In this study we showed that radiation may cause alteration of the neuronal popu- lation types that may lead to or initiate neuronal disorders such as AD. 4.2. X-irradiation of immature neurons alters the dendritic arbors and expression of postsynaptic proteins but not presynaptic proteins at later stages Immature neurons have been shown to contribute to the recovery of neurological function following brain injury in in vivo models (Kernie and Parent, 2010;Villasana et al., 2015). It is essential that immature neurons, even after exposure to radiation, maintain their normal phenotypic development as they mature, including the dendritic arborization, formation of functional synapses and the reliable transmission of information from the presynaptic to the postsynaptic terminal. Here, 0.5 Gy of X irradiation did not impact the dendritic arborization but 1 Gy significantly decreased the number of intersections. While irradiation did not impact expression of the presynaptic protein synapsin 1, the levels of the postsyn- Fig. 8. There are decreases in the number of drebrin clusters 3 weeks after 1 Gy of X-ray exposure. (a) Representative images at DIV 21 for sham, 0.5 Gy and 1 Gy. Immunolabeling for MAP2 (red) and drebrin (green). (b) The number of drebrin clusters along a 100-m length of a dendrite. There was a decrease in the number of drebrin clusters 3 weeks after 1 Gy of irradiation. The difference in the number of clusters between sham and treatment in each irradiated group was analyzed using Student's t-test with Bonferroni correction. **P < 0.01. Each point represents the mean ± SEM for 43-52 different segment from three independent experiments. aptic proteins drebrin and PSD-95 were significantly reduced 3 weeks after irradiation but not at 2 weeks when the number of neurons already decreased. Therefore, irradiation of immature neurons causes changes in the neuronal morphology and synaptic proteins, in the later stage of development, that are essential for synaptic function. Hippocampal synaptic loss is a prominent feature in several neurodegenerative diseases such as Alzheimer Disease, and is associated with cognitive deficits (Counts et al., 2012;Moodley and Chan, 2014). The long-term changes in synap-tic proteins induced by X-irradiation may result in synaptic loss in the brain similar to neurodegenerative diseases. Further studies on the mechanisms underlying radiation-induced delayed cell death and synaptic changes should advance the development of novel therapeutic strategies for treating cognitive impairments caused by radiation exposure during early development.
Our current results strongly suggest that irradiation of developmental stage neurons not only alters the number and population types of remaining neurons after irradiation but also significantly affects postsynaptic proteins such as drebrin and PSD95 when neurons reach maturity and may result in an imbalance of neuronal cell types and synaptic loss in the brain similar to neurodegenerative diseases.

Declaration of Competing Interest
The authors have no conflict of interest to declare.