Radiation Environment and Medicine 2023 Vol.12, No.1 65–73

Regular Article

Effect of Ionizing Radiation on the Intercellular Networkof Murine Cerebral Cortical Neurons
Masaru Yamaguchi1§, Takakiyo Tsujiguchi1§, Tomonori Furukawa 2, Shuji Shimoyama 2,
Toshiya Nakamura3, Yasushi Mariya4 and Junko Yamada5*
Department of Radiation Science, Hirosaki University Graduate School of Health Sciences,66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan2
Department of Neurophysiology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan3
Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences,66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
4
Aomori Rosai Hospital, 1 Shirogane-machi, Hachinohe, Aomori 031-8551, Japan
5
Department of Comprehensive Rehabilitation Science, Hirosaki University Graduate School of Health Sciences,66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
1

Received 13 September 2022; revised 25 November 2022; accepted 7 December 2022

Depending on brain tumor type, radiotherapy with various irradiation methods may be an effective
treatment even for lesions for which surgical treatment is difficult; however, neurocognitive
dysfunction may occur as an adverse effect. Neurons divide infrequently and are recognized
as radiation resistant; however, few reports have investigated the effects of radiation on theintercellular network. We analyzed the impact of radiation exposure on the repair kinetics of DNA
damage and the electrophysiological changes of synaptic currents in cerebral cortical neurons,
employing primary culture technique. Cortical neurons (14 days in vitro) were subjected tovarying X-irradiation doses. DNA double-strand breaks were induced in cultured cortical neurons
by high-dose X-irradiation; however, these cells have the ability to repair severe DNA damage
and are resistant to radiation. An electrophysiological investigation revealed that the inter-event
intervals of miniature excitatory postsynaptic current (mEPSC) in X-irradiated cortical neurons
were significantly longer, while the amplitude showed no change in comparison to 0 Gy-irradiatedcells. These results suggested that the exposure of neurons to radiation led to a decrease in thefrequency of the mEPSC, which affected the synaptic network, which supports the neurocognitive
function despite being less likely to cause cell death or severe DNA damage.
Key words: ionizing radiation, cerebral cortical neurons, electrophysiology, postsynaptic current

1. Introduction*Junko Yamada: Department of Comprehensive Rehabilitation Science Hirosaki
University Graduate School of Health Sciences 66-1 Hon-cho, Hirosaki, Aomori 0368564, Japan
E-mail: jyamada@hirosaki-u.ac.jp§
These authors have made an equivalent contribution to the first author.
https://doi.org/10.51083/radiatenvironmed.12.1_65
Copyright © 2023 by Hirosaki University. All rights reserved.

Malignant neoplasms are the leading cause of death inJapan, accounting for 30% of all deaths1). Radiotherapy
is a topical treatment for cancer. It is considered lessinvasive and capable of preserving the function andmorphology of organs. Radiotherapy is widely used totreat malignant tumors (e.g., primary and metastatic
brain tumors and head/neck cancers) and improves the

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prognosis of these patients2-4); however, radiotherapy
may unavoidably induce injury to the healthy central
nervous system (CNS) surrounding the tumor5-9). Cerebral
necrosis, leukoencephalopathy, and cerebral atrophy are
well known examples of radiation-induced damage to the
CNS10-17). Although classic radiation necrosis has becomeless common, it is increasingly reported that subtle CNS
toxicities (e.g., progressive cognitive dysfunction or
impairment of the neurocognitive function (NCF)) may
occur within the classically “tolerable” radiation dose
range of approximately 20–30 Gy in 10 fractions within
3 weeks8, 18-22). On the other hand, Mariya et al. reported
that neurofunctional recovery was observed in a large
proportion of patients who received radiotherapy forbrain tumors23). In addition, Aoyama et al. demonstrated
that whole brain radiotherapy affected the NCF ofpatients with brain metastases who received stereotactic
radiosurgery, and that brain tumor control is the mostimportant factor for stabilizing the NCF; however, thelong-term adverse effects of whole brain radiotherapy
on the NCF might not be negligible19). The presenceof restoration or stabilization of the CNS function may
reflect plasticity of the residual neurons and their
networks against CNS damage caused by the tumor
itself and radiation exposure24); however, this remains to
be clarified. Although neurons are generally thought tobe resistant to radiation25), there is a scarcity of detailed
information regarding how radiation affects neurons andsynaptic transmission and causes memory impairment.
Hence, to elucidate the genetic damage/repair andelectrophysiological changes in synaptic transmissionthat could underlie the neuroplasticity, we investigated
the impact of radiation exposure on the repair kinetics of
DNA double-strand breaks (DSBs) and serial changes ofsynaptic currents in cerebral cortical neurons, employing
primary culture.
2. Materials and methods
2.1. Ethics statement
All experiments were conducted according to the legal
regulations in Japan and the Guidelines for Animal
Experiments after obtaining approval from the animal
experimental committee (approved number: G140006),
and all efforts were made to minimize the number ofanimals used and their suffering in this study. All mice
were housed in standard cages in a conventional clean
room under a 12-h light/dark cycle. The mice had ad
libitum access to sterilized standard laboratory mousechow diet and drinking water.
2.2. Primary cultures of embryonic mouse cerebral corticalneurons
Primary neuronal cortical cultures were prepared

as described previously 26). Briefly, cerebral cortices
were dissected from C57BL/6JJcl mice (Clea Japan,
Tokyo, Japan) embryos at 15–16 days of gestation (8–10
embryos from 13 dams each). After removal of themeninges, the cortices were cut into small pieces (< 1mm3) in phosphate-buffered saline (PBS). The tissue wassedimented and washed three times with Dulbeccoʼs
modified Eagleʼs medium (Invitrogen, Carlsbad, CA,USA), and then incubated at 37 °C for 10 min in an
enzyme solution containing 2.5 mg/mL trypsin and
0.01% DNase I (Sigma-Aldrich, St. Louis, MO, USA).
After enzymatic digestion and mechanical triturationin serum-free medium, the cells so obtained weresuspended in the medium. Cells were plated on poly-Llysine-coated coverslips in 24-well culture plates at 2.0
×105 cells in 1 mL/well, and maintained in serum-free
medium. The serum-free culture medium consisted of
Neurobasal medium (Invitrogen) supplemented with 2%
B27 supplement (Invitrogen), 0.5 mM L-glutamine, and
100 units/mL penicillin-streptomycin (Invitrogen). Half ofthe volume of the medium was replenished twice a week.
Cells were cultured under these conditions for 14 days invitro (DIV14 cortical neurons) and used for the following
irradiation experiments. The number of viable cells wascounted on day 7 using the trypan blue dye exclusion
method (Sigma-Aldrich) after collected cortical neurons
with 0.1% trypsin-ethylene diamine tetraacetic acid (Gibco;
Thermo Fisher Scientific).
2.3. In vitro irradiation with X-rays
DIV14 cortical neurons in serum-free medium were
subjected to varying X-irradiation doses of 0, 0.1, 0.5, 1, 2,
4 or 7 Gy (150 kVp, 20 mA, 0.5-mm aluminum and 0.3-mm
copper filters) at a dose rate of 1.0 Gy/min using an MBR1520R X-ray generator (Hitachi Medical, Tokyo, Japan) at
a distance of 45 cm between the focus and the target. The
dose was monitored with a thimble ionization chamberthat was placed next to the sample during irradiation anddishes containing DIV14 cortical neurons were arranged
concentrically.
2.4. Measurement of cell deathFor detection of cell death, the cells were first washed twicewith cold PBS and then fixed with 4% paraformaldehyde
for 30 min. The fixed cells were washed again with PBS
and mounted using Vectashield® Mounting Medium with
4ʼ,6-diamidino-2-phenylindole (DAPI, Vector Laboratories,
Burlingame, CA, USA). The cell death was examined
under an Olympus IX71 fluorescent microscope (Tokyo,
Japan) and its DP2-BSWsoftware with a magnification of
40. Cells containing condensed nuclei were considered to
be dead. Data were collected from at least three random
sections per dish.

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2.5. Immunof luorescent detection of DN A DSB repair
related proteins
DIV14 cortical neurons were washed with PBS, and fixedwith ice-cold 75% ethanol for 10 min at room temperature.
Fixed cells were washed with PBS, permeabilized in
0.5% Triton X-100 (Wako, Osaka, Japan) on ice for 5min, and washed twice with PBS. The cells were thenincubated with an anti-phospho-histone H2AX (ser139)
mouse immunoglobulin G monoclonal antibody and anti53BP1 mouse immunoglobulin M monoclonal antibody(Millipore, Billerica, MA, USA) diluted 1:300-fold with
TBST (20 mM Tris-HCl [pH 7.4], 137 mM NaCl, 0.1%
TWEEN-20) containing 5% skim milk at 37 °C for 120
min, and subsequently washed with PBS and incubatedwith Alexa Fluor 488 goat anti-mouse immunoglobulin
G (H+L) antibody and Alexa Fluor 546 goat anti-mouse
immunoglobulin M (μ chain) antibody diluted 1:400fold with TBST containing 5% skim milk at 37 °C for
60 min. Following a second wash with PBS, the cells
were adhered to microscope glass slides (Matsunami
Glass, Osaka, Japan) using a StatSpin® CytoFuge 2 (Iris
Sample Processing, Westwood, MA, USA) and mounted
using Vectashield ® Mounting Medium with DAPI.
For the quantitative analysis, the phosphorylation of
the histone variant H2AX at serine 139 ( γ -H2AX) andtumour suppressor p53 binding protein1 (53BP1) fociwere counted per cell using a Laser Scanning Microscope
710 (Carl Zeiss Microscopy, Tokyo, Japan) with amagnification of 6327, 28). Under blinded conditions, thenumbers of γ -H2AX and 53BP1 foci per cell were counted
for more than 50 cells in every sample.
2.6. Electrophysiology
As we reported in previous papers26, 29), the synaptic
currents were measured by voltage-clamp and patchcla mp met hod t o a na l yse cha nges in neurona ltransmission. DIV14 cortical neurons were transferred to arecording chamber on the stage of an upright microscope
ECLIPSE E600FN (Nikon, Tokyo, Japan), continuously
perfused with artificial cerebrospinal fluid (ACSF) at aflow rate of 1−2 mL/min at 31−32 °C , and viewed on amonitor with infrared differential interference contrast.
The standard ACSF contained 126 mM sodium chloride,
2.5 mM potassium chloride, 1.25 mM sodium dihydrogen
phosphate, 2.0 mM magnesium sulfate, 2.0 mM calcium
chloride, 26.0 mM sodium bicarbonate and 20.0 mM
glucose with 95% O2 and 5% CO2. The resting membranepotential and membrane resistance were recorded before
miniature excitatory postsynaptic current (mEPSC)
without tetrodotoxin (TTX). mEPSC recordings wereobtained in whole cell voltage-clamp mode with holding
potential at -70 mV in the presence of 1 µM tetrodotoxin.
Patch electrodes had 4−5 MΩ resistance when filledwith a solution containing 150 mM potassium methane67

sulfonate, 5 mM potassium chloride, 0.5 mM ethylene
glycol-bis (2-aminoethylether)-N,N,Nʼ,Nʼ-tetra acetic acid,
10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid, 3 mM magnesium (adenosine triphosphate)2, and
0.4 mM guanosine triphosphate; pH 7.3. Patch electrodes
were fabricated from borosilicate capillary tubing of 1.5
mm in diameter (Garner Glass, Claremont, CA, USA)using a Narishige PP-83 vertical puller (Narishige, Tokyo,
Japan). Recordings were made with a MultiClamp 700B
amplifier (Molecular Devices, CA, USA) in conjunction
with a Digidata 1322A digitizer (Molecular Devices) andthe pClamp 10 software suite for the Clampex software(Molecular Devices). All signals were filtered at 2 kHz
and sampled at 10 kHz. mEPSCs were recorded at least
3 min. Averaged values of all cells were showed in every
30 s for amplitude and inter-event intervals of mEPSCs
analyzed using the Mini Analysis program (Synapto soft,USA) with a threshold of 6 pA for event detection.
2.7. Statistical analyses
Significance levels were calculated using the Origin
software package (Origin Lab, Northampton, MA, USA)for the Windows operating system. The t test was used
for the comparisons between two groups, while the Steeltest was used for the comparisons among multiple groups.
P values of 0.05 were considered to indicate statisticallysignificant differences.
3. Results
3.1. Cell viability and repair kinetics of DNA DSB in murine
cerebral cortical neurons
In order to investigate the response of DIV14 corticalneurons to high dose of X-rays in this study, the numberof viable cells was counted one week later using thetrypan blue dye exclusion method and DAPI nuclear
staining. As shown in Fig. 1A, almost cortical neurons
subjected to X-irradiation doses of 0 Gy (left panel) or
7 Gy (right panel) were not shrunken. Cortical neurons
retained the ability to grow after exposure to 7 Gy,and no statistically significant differences in viable cellnumbers were observed between non-irradiated and 7
Gy-irradiated cells (P = 0.6176, Fig. 1B). Additionally,
we next compared X-irradiation-induced cell death (Fig.
1C). DAPI nuclear staining observed by fluorescencemicroscopy showed normal nuclear labeling as very
diffuse large intact structures, rather than bright small
bodies which is a typical feature of nuclear condensation.
The number of apoptotic cells labeled by DAPI did notappear to increase after X-irradiation, relative to control.
No significant effects on cell viability were detected
between non-irradiated (75.6±9.4%) and 7 Gy-irradiatedcells (72.3±5.4%, P = 0.3140, Fig. 1D). These data indicatethat the cytotoxicity of X-rays was not severe in immature

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A

B

C

D

Fig. 1. Cell viability assessment of X-irradiated DIV14 cortical neurons. [A] Representative photographs of cultured cortical neurons
at one week after X-irradiation are shown in the left (0 Gy) and right panel (7 Gy) (10×magnifications, scale bar 100 μm). [B] Viable
cells in each condition were counted using the trypan blue dye exclusion method and the surviving fraction was calculated basedon initial number of seeded cells (2.0×105 cells/ml). The data are expressed as the mean±standard deviation (SD) of 3 independent
experiments. [C] Representative photomicrographs of DAPI-stained nuclei of cortical neurons are shown in the left (0 Gy) and rightpanel (7 Gy) (40×magnifications, scale bar 10 μm). Yellow arrow indicates radiation-induced nuclear condensation considered to bedead. [D] Cell viability relative to total cell count in at least three random sections per dish (n = 12 in each condition). The data areexpressed as the mean±standard error (SE). No statistically significant differences were observed between two groups by t test.

neurons. In order to evaluate the DNA DSB formation
and repair kinetics of DIV14 cortical neurons, the fociof γ -H2AX, a DNA DSB marker, and the DNA damage
response protein 53BP1, which interacts with γ -H2AX
to co-localize to the DSB site and accurately repair DNA
damage, were analyzed in these cells subjected to varying
X-irradiation doses of 0, 0.1, 0.5, 1, 2, or 4 Gy and harvested
from 0.5 h to 168 h post-irradiation, and a representative
image is shown in Figure 2A. A radiation-dose dependent
increase in the numbers of γ -H2AX and 53BP1 foci after
X-irradiation was observed in cortical neurons. In 0.1−
4 Gy-irradiated cells, the number of γ -H2AX foci peaked
at 0.5−1 h after X-irradiation, and was approximately 10−68 fold higher than that in non-irradiated cells (Fig. 2B,
Table 1). Similar to the results observed in the γ -H2AXexpression, the number of 53BP1 foci of 0.1−4 Gyirradiated cortical neurons was approximately 10−60 foldhigher than that of non-irradiated cells at 0.5−1 h after
X-irradiation (Fig. 2C, Table 1). Although the γ -H2AX
and 53BP1 foci per nucleus of the irradiated cells began togradually decrease after 1 h, the numbers of residual fociof γ -H2AX and 53BP1 in the 2−4 Gy-irradiated corticalneurons were still significantly higher (2−15 fold and 6−
14 fold, respectively) in comparison to non-irradiated cells

at 12 and 24 h after X-irradiation. After 48 h have passed,
the numbers of γ -H2AX and 53BP1 foci decreased tothe same level as in non-irradiated cells. These results
suggested that DNA DSB were induced in cultured
DIV14 cortical neurons by X-irradiation; however, thesecells have the ability to repair severe DNA damage andare resistant to radiation.
3.2. E f f ect of X-irradiation exposure on the synaptic
responses of murine cerebral cortical neurons
First, we observed the membrane property of each group.
Before applied TTX, cortical neurons were recorded theresting membrane potential and resistance by current
clamp mode. There were no significant differences inall groups (Fig. 3A and 3B). Neurons and their synapses
are the basic building blocks of a complex giant networkof the brain, and transmitted information is coded by
the timing and number of action potentials, which is
generated by the opening of voltage-gated sodium
channels during depolarization. For analyzing of theeffect of irradiation on the synapse, we used mEPSCs,
which indicates the single quantum synaptic responses30).
These mEPSCs can be detected during total blockade ofaction potentials by TTX. In order to clarify the effects ofMasaru Yamaguchi et al. / Radiation Environment and Medicine 2023 Vol.12, No.1 65–73

A

69

B

C

Fig. 2. DNA DSB formation and repair kinetics. [A] Representative co-localized images of γ -H2AX and 53BP1 in cortical neurons
obtained 0.5 h, 3 h, and 168 h after X-irradiation with 0.1−4 Gy. Nuclei, γ -H2AX foci, and 53BP1 foci are shown in blue, green, andred, respectively (63×magnifications, scale bar 5 μm). [B] γ -H2AX and [C] 53BP1 foci were counted in each cell. Data are expressedas the mean±SD of 3 independent experiments for each dose and time point. A Steel multiple comparison test revealed statisticallysignificant differences among cells irradiated with 0 Gy to 4 Gy of X-rays and statistical details were summarized in Table 1.

Table 1. Summary of fold-change (FC) with P value in DNA double-strand break repair kinetics.
hrs
0.5 h

Gy
FC
P value

γ -H2AX foci number normalized by 0 Gy
0.1 Gy0.5 Gy
1 Gy
2 Gy
4.0
4.7
20.2
26.5
0.03700.0111< .0001< .0001

4 Gy
35.0< .0001

0.1 Gy3.4-

53BP1 foci number normalized by 0 Gy
0.5 Gy
1 Gy
2 Gy
4 Gy
4.7
15.9
31.4
28.9
0.0154< .0001< .0001< .0001

FC
10.7
28.1
52.4
65.1
68.3
10.1
25.9
45.8
54.3
59.6
P value< .0001< .0001< .0001< .0001
0.0148< .0001< .0001< .0001< .0001
FC
5.1
17.2
29.6
28.9
39.6
4.8
17.6
31.6
30.5
40.3
3h
P value< .0001< .0001< .0001< .0001< .0001< .0001< .0001< .0001
FC
4.1
9.7
8.3
10.4
50.3
3.3
8.4
4.9
11.3
39.7
6h
P value< .0001
0.0002< .0001< .0001
0.00010.0450< .0001< .0001
FC
3.5
3.9
2.1
11.8
15.2
2.8
3.1
1.9
11.6
12.0
12 h
P value< .0001< .0001< .0001< .0001
FC
0.5
1.7
4.7
6.3
2.8
0.4
3.0
10.0
14.0
6.0
24 h
P value< .0001< .0001
0.00020.00090.01040.0104
FC
1.3
2.1
2.0
1.5
0.9
0.9
2.6
2.1
1.9
1.3
48 h
P value
FC
1.7
1.4
0.7
2.2
0.6
1.8
1.4
0.8
2.0
0.7
168 h
P value
Note. FC shows that the average foci number of γ -H2AX or 53BP1 at each dose is normalized by that of the 0 Gy. No value “ - ” means there wasno statistically significant difference (P > 0.05). P values of 10-5 or less were unified as “< .0001”.
1h

X-ray exposure on the glutamatergic excitatory synapses,
representative images of DIV14 cortical neurons weretaken 2−3 days after X-irradiation with 0 Gy, 2 Gy, 4 Gy,
and 7 Gy using whole-cell patch-clamp recordings. The
mEPSC waveforms are shown below each representative
image, and the vertical and horizontal directions of the
waveform indicate the amplitude and the frequency,
respectively (Fig. 3C). The amplitude reflects thesensitivity to transmitters in the postsynaptic membranes
and the frequency (inter-event intervals) reflects theprobability of transmitter release from the synaptic
terminal. Although no significant effects on the amplitudeof mEPSC were observed in cortical neurons subjected to
X-irradiation doses of up to 7 Gy (Fig. 3D), the inter-event
intervals of mEPSC in 4 Gy-irradiated cortical neurons(235.2±42.3 msec, P = 0.0093) and 7 Gy-irradiated cortical

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A

C

B

D

E

Fig. 3. Membrane properties and alterations in mEPSC. [A] The mean resting membrane potential and [B] membrane resistancemeasured by current injection in 0−7 Gy (n = 6 ~ 8 each). [C] Representative images and recordings of cortical neurons taken 2−3days after X-irradiation with 0−7 Gy. Scale bar, 10 μm. The mEPSC tracing is shown below the cell image. The effect of X-irradiation
on [D] mEPSC amplitude (pA) and [E] inter-event interval (msec). Error bars represent the mean ± SE in 0 Gy (n = 31, 16 plates), 2
Gy (n = 16, 8 plates), 4 Gy (n = 19, 10 plates), and 7 Gy (n = 15, 8 plates) irradiated groups, respectively. The statistical significance ofdifferences among 0 Gy-, 2 Gy-, 4 Gy-, and 7 Gy-irradiated groups was evaluated by the Steel multiple comparison test.

neurons (291.5±94.5 msec, P = 0.0001) were significantly
longer in comparison to non-irradiated cells (77.8±
9.0 msec) (Fig. 3E), suggesting that radiation exposure
reduces the frequency of the mEPSC and causes a
decrease in the release of transmitters from the synaptic
terminal.
4. Discussion
The aim of this study was to investigate the effect ofradiation exposure on the repair kinetics of DNA DSB andthe serial changes in the synaptic network in embryonicmurine cerebral cortical neurons, employing a primaryculture technique. This useful technique has alreadybeen reported in several papers in order to elucidate theneurotoxic action of organotin compounds and to clarify
the activation mechanism of K+ Cl– co-transporter function
by brain-type creatinine kinase, suggesting that an in vitro
model of dissociated cortical neurons recapitulates theconnectivity within the intact cortex26, 31). Furthermore,morphological and electrophysiological changes inintratelencephalic-type pyramidal neurons in the motor
cortex of a rat model of levodopa-induced dyskinesia were
investigated, which shows how abnormal transmission
between synapses impairs central nervous system
function29). Neural stem cells and vascular endothelial
cells are generally radioresistant; however, these cellulardisorders cause the suppression of neurogenesis, a
progressive reduction in the survival of clonogenic cells,
ischemic changes, and the break-down of the blood brain
barrier, which causes regression and the deterioration of
the neuronal network and function8, 32-35). For both patients
undergone radiation for brain tumors and their caregivers,
the distressing adverse effect is impaired cognition.
This debilitating effect have suffered from inadequate
understanding of the cellular mechanisms of radiation
damage, and little information exists on the acute effectsof radiation on synaptic function. In this study, after 48
h passed, DNA DSB formation, which was monitored byimmunocytochemistry of γ -H2AX and 53BP1, returned tothe baseline levels of non-irradiated cells, and the corticalneurons used in this study were found to have the ability
to repair severe DNA damage and to be resistant to highdose irradiation (Fig. 1 and Fig. 2). On the other hand, no
significant effects on the amplitude of mEPSC, sensitivity
to transmitters in the postsynaptic membranes, wasobserved in cortical neurons subjected to X-irradiation
doses of up to 7 Gy (Fig. 3D). However, the interevent intervals of mEPSC, reflecting the probability oftransmitter release from the synaptic terminal, in 4 Gy
and 7 Gy-irradiated cortical neurons were significantly
longer in comparison to non-irradiated cells (Fig. 3E).
Radiation exposure reduces the frequency of mEPSC;
thus, decreasing the release of transmitters from theMasaru Yamaguchi et al. / Radiation Environment and Medicine 2023 Vol.12, No.1 65–73

synaptic terminal. However, the lack of a significant effecton the amplitude of mEPSC implies that the susceptibilityof transmitter receptors at the postsynapse was unaffectedby radiation exposure. In other words, radiation exposure
presynaptically suppressed the synaptic transmission ofneurons, suggesting that the effects of radiation exposure
appear at the presynaptic terminals. Furthermore, thefrequency of mEPSC depends on the density changeof the dendritic spines on the postsynaptic cell, and thesignal intensity is considered to depend on the post-sidechange and/or the spine size36). It is reported that thespine form changes during conditions such as mental
retardation and memory impairment, and changes in
conjunction with learning ability, the ovulatory cycle,
spine shape and spine number are correlated with thesynaptic transmission efficiency37-40). It is also known that
irradiation with X-rays and photons, which has a higher
biological effect ratio in comparison to X-rays, affects
the ability to acquire molecular biological memory41,42).
Another previous study has also assessed the impactof ionizing irradiation on neuronal intrinsic properties
and synaptic signaling. Radiation led to early decreases
in tyrosine phosphorylation and removal of excitatory
N-methyl-D-aspartate receptors from the cell surface
while simultaneously increasing the surface expressionof inhibitory gamma-aminobutyric acid receptors43). Dey
et al. found that serotonin receptor levels were decreasedin the hippocampus post-irradiation, and mice displayed
both anxiety- and depression-like behaviors44). Newton etal. revealed by proteomic analysis the dysregulation of
metabolic and signaling pathways, such as mitochondrial
dysfunction, Rac 1 signaling, and synaptogenesis
signaling, associated with neurocognitive dysfunction45).
These alterations in cellular localization, receptor, or
activity of signaling transduction corresponded withaltered synaptic responses and inhibition of long-term
potentiation. Furthermore, Shirai et al. demonstrated that
X-ray irradiation decreases the dendritic spine density ina dose-dependent manner and changes the dendritic spine
morphology thinner and longer than the non-irradiatedspines by reducing the abundance of cytoskeletal
proteins2). In addition, changes to the irradiated brain
include depletion of stem cell populations accompanied by
reductions in dendritic complexity and spine density, andradiation exposure initiates cyclical cascades of secondary
reactive processes that involve oxidative stress andinflammation, which serve to perpetuate the signatureof radiation injury over time45,46). In consideration of
our result and the findings of various reports, althoughneurons have the ability to repair severe DNA damage,even when irradiated with high doses of X-rays, there isa high possibility that some change, such as density ormorphology, has occurred in the dendritic spines whichhave essential functions in synaptic transmission.

71

The present study revealed that neuron has highresistance for irradiation but has an influence on
the synaptic transmission. Our data showed that thefrequency of mEPSCs was increased, at least in part bysome morphological changes and molecular biologicalchanges might occur in the dendritic spines. Radiotherapy
has been widely used in the treatment of brain tumors;
however, cognitive impairment due to radiation exposure
has been drawing attention as the number of patients
who achieve long-term survival after radiotherapy
increases47-49). Only concomitant adverse events may be
noticeable, especially if the tumor has low proliferative
potential50). Therefore, it is very important to elucidate
the mechanism underlying the occurrence of adverse
events, estimate the degree of cognitive impairment, andpromote efforts to improve the prognosis. Further studies
should be undertaken to analyze the relationship between
radiation exposure and the morphological changes ofspines, as well as the mechanisms of the individual effectsof whole brain irradiation, such as the effects on memory
and behavior.
Acknowledgments
This work was supported by Hirosaki University
Institutional Research Grant FY2014-2016.
Conflict of interests
The authors have no potential conflicts of interest to
declare.
Author contributions
TN, YM and JY designed the study; MY, TT, TF, SS andJY performed the experiments; MY, TT, TN, YM and JY
wrote this manuscript. All authors contributed extensively
to discussions regarding the work and the review of themanuscript.
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