ORIGINAL ARTICLE

Effects of electromagnetic fields on Reelin and Dab1 expressionin the developing cerebral cortex

Matin Hemmati • Farhad Mashayekhi •

Fareheh Firouzi • Masoumeh Ashori •

Hamidreza Mashayekhi

Received: 14 December 2013 / Accepted: 13 February 2014

� Springer-Verlag Italia 2014

Abstract Many studies describe the biological effects of

electromagnetic fields (EMF) including brain damages,

neuronal migration and neurogenesis within the central

nervous system. Neuronal cell produced in the neuroepi-

thelium migrates along radial glial fibers into the cortical

plate. Reelin, which is produced by Cajal–Retzius cells

directs neuronal migration. It was shown that Disabled 1

(Dab1) functions downstream of reelin signal transduction

pathway that directs the correct cytoarchitecture of the

developing cortex. In this study, the EMF effects on total

protein concentration (TPC), reelin and Dab1 expression in

the developing cortex was studied. 30 pregnant Balb/c mice

were separated into three groups: control (n = 10), EMF

(n = 10) and SHAM groups (n = 10). The 15-day preg-

nant mice were placed inside the solenoid for a daily EMF

exposure of 5 h for 3 consecutive days (15–17). The

SHAM group was also located in the same coil with no

exposure. Mice were sacrificed 24 h after the final expo-

sure session. TPC, reelin and Dab1 expression were studied

by Bio-Rad protein assay and western blot. No significant

change in the TPC was seen in the EMF-treated cerebral

cortex samples compared with those from the SHAM and

control groups. It was also shown that the reelin and Dab1

expression increases in the EMF-treated cerebral cortex

extracts as compared to controls and SHAM group. It is

concluded that EMF may play important role in the neural

cell migration by increasing reelin and Dab1 expression in

the developing cortex.

Keywords Electromagnetic fields � Reelin � Dab1 �Cerebral cortex � Development

Introduction

The cerebral cortex develops from two lateral telencephalic

vesicles by successive growth, cell proliferation and

migration from the germinal epithelium. Neurons and glia

produced in the germinal epithelium migrate along radial

glial fibers through the subplate and into the cortical plate.

They migrate past previously laid down neuronal layers to

reach the Cajal–Retzius (CR) cells of the marginal zone

[1]. The CR cells express the microtubule-associated pro-

tein-2, which is a marker for neuron [2]. CR cells contain a

variety of calcium-binding proteins, such as calbindin,

calretinin and parvalbumin [3]. CR cells express extracel-

lular matrix proteins, which are involved in migration.

They also secrete reelin, which is important in the correct

lamination of the cerebral cortex during development [4].

Reelin is an extracellular 420-kDa glycoprotein that

binds to the transmembrane receptors apolipoprotein

receptor 2 and very-low density lipoprotein receptor

(VLDLR), which transduce the reelin signal through the

disabled-1 (Dab-1) [5]. Reelin signaling induces tyrosine

phosphorylation of Dab1 triggering a tyrosine kinase cas-

cade that ultimately controls proper neuronal migration and

positioning during the central nervous system (CNS)

development [6]. Reelin’s function in the adult brain is far

less well understood, but altered brain and blood reelin

levels have been reported in some neurological disorders

[7] and the possibility has been considered as an

M. Hemmati � F. Mashayekhi (&) � F. Firouzi � M. Ashori

Department of Biology, Faculty of Sciences,

University of Guilan, Rasht, Iran

e-mail: umistbiology@yahoo.co.uk; mashayekhi@guilan.ac.ir

H. Mashayekhi

Department of Physics, Faculty of Sciences,

University of Guilan, Rasht, Iran

123

Neurol Sci

DOI 10.1007/s10072-014-1690-z

involvement of the reelin signaling pathway in neurode-

generative disorders [8].

Reelin is thought to play its role as a guide for migratory

neurons via the interaction with two cell surface receptors,

the VLDLR and the apolipoprotein E receptor 2, then

triggering a tyrosine kinase-signaling cascade [9].

It has been demonstrated that Dab1 functions down-

stream of reelin in a lysine kinase signal transduction

pathway that controls appropriate cell positioning in the

developing brain. Dab1 is a cytosolic protein that activates

tyrosine kinases. The response of cortical plate cells to

reelin requires the tyrosine kinase adaptor Dab1 [10]. Dab1

signaling instructs post-migratory neurons to detach from

their radial guides, and form a dense, well-organized cor-

tical plate. It was shown that hepatocyte growth factor is

essential for reelin and Dab1 expression in the cerebral

cortex [11].

Effective directional neuron migration is crucial in

development of the CNS and for neurogenesis. Endoge-

nous electrical signals are present in many developing

systems and crucial cellular behavior such as neuronal cell

division, cell migration and cell differentiation are all

under the influence of such endogenous electrical cues

[12]. It has been suggested that EMFs might interact with

the local geomagnetic field to affect cell migration in

structures within the brain stem [13]. In this study, we

examined the effects of EMF in reelin and Dab1 expression

in the developing cerebral cortex.

Materials and methods

Animals

Balb/c mice were purchased from Pastor Institute, Tehran,

Iran and maintained on 12–12 light:dark cycle beginning at

8.00 a.m. They were kept in the cages 30 9 40 9 40 cm

(W 9 L 9 H) at a constant temperature in mice boxes with

unrestricted access to laboratory food pellets (Pars Com-

pany, Tehran, Iran) and water. The colony was maintained

through random pair mating. Timed mating was carried out

by placing a male and female together and checking for the

presence of a vaginal plug. The presence of a vaginal plug

was taken as gestational day 0 (E0) and the day of birth was

designated postnatal day 0 (P0). All animal procedures

were carried out in accordance with the Animals (Scientific

Procedure) Act, 1986.

Design and description of EMF emitter set

The solenoid (EMF producer) was designed for producing

EMF with 50 Hz frequency and (0.5 mT) intensity. An

urban electric line adaptor 220 V 10 A was used for

minimizing the heat production by EMF emitter coin. The

EMF emitter set including bobbin (80 9 10), wires and

metal nucleuses was put in the bottom of hatchery machine

in a metal lacuna. The calibration of the exposure facility

was carried to confirm uniform distribution of the magnetic

field intensity where mice were kept.

EMF exposure

Thirty pregnant Balb/c mice on day 15 of gestation were

separated into three groups: control (n = 10), EMF

(n = 10) and SHAM groups (n = 10). Three independent

exposures/sham exposures were performed. They were

placed inside the solenoid for a daily EMF exposure of

approximately 1 mT (50 Hz magnetic field) of 5 h for 3

consecutive days (15–17). The control group was not

subjected to any procedure. The SHAM group was also

located in the same coil with no exposure. Mice were killed

24 h after the final exposure session on day 18 of gestation

after killing of the mother, by excessive dose of anesthetic

(sodium pentobarbitone), at gestation day 18 to the effects

of EMF on reelin and Dab1 expression in the developing

cerebral cortex. Fetuses were decapitated and the cerebral

cortex were removed and processed as described below. A

total number of 156 fetuses were collected from the all

pregnant mice [EMF exposed (n = 56), SHAM (n = 48)

and control (n = 42)]. The temperature and humidity were

monitored continuously throughout the experimental per-

iod. This ensures that the control and the exposed animals

were maintained in the same condition. The experiences

were carried out under a blind condition.

Cell extract

Tissue (cerebral cortex) samples (10 mg each) were

chopped into tiny pieces and suspended in 0.5-ml protein

lysis buffer [150 mM NaCl, 1.0 % NP40, 20 mM Tris (pH

7.5), 5 mM EDTA, and Complete Mini protease inhibitor

cocktail (Roche Diagnostics, West Sussex, UK)] and then

mechanically homogenized by sonication. After centrifu-

gation, the protein extracts were recovered and stored at

-70 �C until they were used.

Total protein concentration and Western blotting

The total protein concentration in cerebral cortex extracts

was determined by the Bio-Rad protein assay based on the

Bradford dye procedure. For Western blot, protein extracts

(50 lg/lane) were separated on 10 % SDS–polyacrylamide

gel and transferred to a polyvinylidene difluoride mem-

brane (Bio-Rad Laboratories Ltd. Hertfordshire, UK). The

membranes were blocked with phosphate-buffered saline

containing 0.05 % Tween 20 and 5 % dry milk and probed

Neurol Sci

123

either with monoclonal mouse anti-reelin [G10] (ab 18570)

and Dab1 (ab 16674) antibodies (Abcam, Cambridge, UK)

(1:1,000 dilution) or a mouse monoclonal anti-b-Tubulin

antibody (loading control) (Abcam plc, Cambridge, UK)

(1:10,000 dilution) and then treated with the appropriate

horseradish peroxidase-conjugated secondary antibodies.

Immunoreactive protein was visualized using the Enhanced

Chemiluminescence Western blotting detection system

(Amersham Pharmacia Biotech, Piscataway, NJ). Densi-

tometric analysis was performed by scanning immunoblots

and quantitating protein bands using an image analyzer

(Metaview Software, version 4.0, UK).

Statistical analysis

All data presented are expressed as mean ± standard error

of the mean (SEM). Statistical analysis was performed

using the one-way ANOVA to test for differences among

the groups, and only values with P B 0.05 were considered

as significant.

Results

Total protein concentration

The total protein concentration in the cerebral cortex

extracts from EMF-exposed, SHAM and control group was

determined by the Bio-Rad protein assay based on the

Bradford dye mixture. The total protein contents of EMF-

exposed, SHAM and control were 0.93 ± 0.07, 0.91 ± 0.1

and 0.90 ± 0.09 (g/l), respectively. No significant increase

in the total protein concentration was seen in the EMF-

exposed cerebral cortex samples compared with those from

the SHAM and control groups (P [ 0.05) (Fig 1).

Analysis of reelin and Dab1 expression

Western blot analysis was performed to quantitatively

evaluate reelin and Dab1 expression in the cerebral cortical

extracts. A Western blot analysis using anti-reelin and anti-

Dab1 antibodies as a probe confirmed the presence of re-

elin and Dab1 in all the extracts (Figs. 2a, 3a). An image

analyzer was used to determine the intensities of the band

in the respective lanes. Quantification of the Western blot

bands from repeated experiments showed that the expres-

sion of reelin and Dab1 was significantly increased in the

EMF-treated cerebral cortical extracts when compared with

SHAM and control groups (P \ 0.0001). However, no

significant changes in the Dab1 and reelin expression in the

mouse cerebral cortex were seen between SHAM and

control groups (P [ 0.05) (Figs. 2b, 3b).

Discussion

Life on earth originated in the natural magnetic fields, but a

large part of living matter became subjected to artificial

Total protein concentration in the cerebral cortex

0

0,2

0,4

0,6

0,8

1

Groups

g/l

Control

SHAM

EMF treated

Fig. 1 Total concentration of protein in the cerebral cortex from

control, EMF-treated and SHAM groups (g/L). No significant

difference was seen in total protein concentration between the

groups. Error bars indicate the standard error of the mean for the

number (n) of 42, 48 and 56 fetuses for control, SHAM and EMF

groups, respectively

AReelin

-tubulin

1 2 3 4

Relative Reelin expression in the cerebral cortex

0

2

4

6

8

10

12

14

16

Groups

Rel

ativ

e ex

pre

ssio

n

Control

SHAM

EMF

B

Fig. 2 a Expression of reelin in the cerebral cortex from control

(lane 1), SHAM- (lane 3) and EMF-treated (lanes 2 and 4) mouse

embryos. b-Tubulin (50 kDa) expression was determined as a protein-

loading control. b Relative reelin expression. Signal intensities from

control- and EMF-treated immunoblotting experiments were deter-

mined by densitometric analysis. The bars represent standard error of

the mean. Significant difference in the reelin expression has been seen

in the EMF-treated cerebral cortex extracts when compared with

SHAM and control groups (P \ 0.001). No significant difference in

the reelin expression was seen between SHAM and control groups

(P = 0.3)

Neurol Sci

123

EMF only during the last century. The first epidemiological

correlation between EMF and the onset of human diseases

was discovered by Wertheimer and Leeper who found that

children living near the electro duct in Denver had an

increased risk of developing leukemia [14]. It has been

demonstrated that EMF negatively affects early fertility

outcome [15]. EMF may act as a promoter of cancer [16].

In addition, many other pathologies were suspected to be

correlated with EMF exposure, such as neurodegenerative

diseases, teratogenicity and infertility [17–19]. The bio-

effects of ELF and RF fields are very different. The results

indicate that the magnetic field induced by a 20 or 60 Hz

has an enhancing effect on the early growth of mung beans,

but the magnetic fields induced by other frequencies (30,

40, and 50 Hz) have an inhibitory effect, especially at

50 Hz [20].

There has been considerable recent progress in under-

standing the processes involved in cerebral cortical devel-

opment. In the developing nervous system, multipotential

stem cells give rise to neurons and glia. Normal develop-

ment of the cerebral cortex depends on the correct prolif-

eration of the stem and progenitor cells and successive

differentiation and migration of cells born in the germinal

epithelium [21]. Neurons and glia produced in the

neuroepithelium migrate along radial glial fibers through

the subplate and into the cortical plate [22].

In this study, we showed that reelin and Dab1 expression

in the developing mouse cerebral cortex is increased in

response to EMF. Coordinated migration of neurons in the

developing and adult brain is essential for its proper

function. Reelin regulates radial glial migration of the

neurons in the developing cerebral cortex by inducing

tyrosine phosphorylation of an intercellular adaptor pro-

tein, Dab1. Dab is essential component of reelin pathway

[23]. In the reelin-deficient mutant reeler, cortical lamina-

tion is inverted with many neurons invading the marginal

zone and others that are unable to migrate to their desti-

nation, suggesting a role for reelin signaling in dynamic

cytoskeletal reorganization. It has been suggested that re-

elin may act as a stop signal for radially migrating neurons

[24].

The results of this study suggest that EMF increases

reelin and Dab1 expression in the developing cerebral

cortex. As reelin and Dab1 play an important role in the

neural cell migration, thus, it is concluded that EMF may

affect on the neural cell migration by altering reelin and

Dab1 expression in the developing cerebral cortex.

Acknowledgments This study was supported by the University of

Guilan. The authors thank all people in the Genetics and Develop-

mental Biology laboratories, Department of Biology, Faculty of

Sciences, University of Guilan, for their technical assistance.

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Relative Dab 1 expression in the cerebral cortex

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