Wireless-Protection.org

Upsala Journal of Medical Sciences. 2010; 115: 91–96
ORIGINAL ARTICLE
Effect of radio-frequency electromagnetic radiations (RF-EMR)
on passive avoidance behaviour and hippocampal morphology in
Wistar rats
SAREESH NADUVIL NARAYANAN1, RAJU SURESH KUMAR1,
BHAGATH KUMAR POTU2, SATHEESHA NAYAK3,
P. GOPALAKRISHNA BHAT4 & MANEESH MAILANKOT5
1Department of Physiology, Melaka Manipal Medical College, Manipal University, Manipal, India, 2Department of
Anatomy, Kasturba Medical College, Manipal University, Manipal, India, 3Department of Anatomy, Melaka Manipal
Medical College, Manipal University, Manipal, India, 4Department of Biochemistry, Kasturba Medical College, Manipal
University, Manipal, India, and 5Department of Biochemistry, Melaka Manipal Medical College, Manipal University,
Manipal, India
Abstract
Introduction. The interaction of mobile phone radio-frequency electromagnetic radiation (RF-EMR) with the brain is a serious
concern of our society.
Objective. We evaluated the effect of RF-EMR from mobile phones on passive avoidance behaviour and hippocampal
morphology in rats.
Materials and methods. Healthy male albino Wistar rats were exposed to RF-EMR by giving 50 missed calls (within 1 hour) per
day for 4 weeks, keeping a GSM (0.9 GHz/1.8 GHz) mobile phone in vibratory mode (no ring tone) in the cage. After the
experimental period, passive avoidance behaviour and hippocampal morphology were studied.
Results. Passive avoidance behaviour was significantly affected in mobile phone RF-EMR-exposed rats demonstrated as shorter
entrance latency to the dark compartment when compared to the control rats. Marked morphological changes were also
observed in the CA3 region of the hippocampus of the mobile phone-exposed rats in comparison to the control rats.
Conclusion. Mobile phone RF-EMR exposure significantly altered the passive avoidance behaviour and hippocampal
morphology in rats.
Key words: Hippocampus, memory, mobile phone, passive avoidance, RF-EMR (radio-frequency electromagnetic radiation)
Introduction
The use of mobile phones is increasing day by day,
and it is estimated that approximately 500 million
people worldwide are using mobile phones currently.
A large proportion of users is made up of children and
teenagers. Due to the wide and growing use of mobile
communication, there is increasing concern about
the interactions of electromagnetic radiation with
the human organs and, in particular, with the brain.
Experimental studies have shown that the radiofrequency
electromagnetic radiation (RF-EMR)
emitted from the mobile phones can affect the brain
in various ways. These effects have been described
in vitro and in vivo in a number of studies: in particular,
effects on cerebral blood flow (1–4), blood-brain barrier
permeability (4), oxidant and antioxidant balance
(5), neurotransmitter balance (6), nerve cell damage
(7), and genomic responses (8) have been reported.
There is some concern that short-term memory loss
Correspondence: Sareesh Naduvil Narayanan, Department of Physiology, Melaka Manipal Medical College, Manipal University, Manipal 576104, India.
E-mail: sareeshnn@yahoo.co.in
(Received 3 August 2009; accepted 10 December 2009)
ISSN 0300-9734 print/ISSN 2000-1967 online 2010 Informa UK Ltd. (Informa Healthcare, Taylor & Francis AS)
DOI: 10.3109/03009730903552661
or other cognitive effects may be associated with
the use of mobile telephones. In our previous study
we had reported that mobile phone exposure in
Wistar rats resulted in impaired spatial memory
performance in the Morris Water Maze (MWM)
test, demonstrated as more time taken to reach the
target quadrant and less time spent in the target quadrant
(9). In the present study, we tried to evaluate the
effect of long-term exposure to RF-EMR emitted from
a mobile phone (0.9 GHz/1.8 GHz) on passive avoidance
behaviour and hippocampal morphology in male
Wistar rats.
Materials and methods
Animals
Inbred healthy male albino Wistar rats (8–10 weeks
old) were used in this experiment. They were
obtained from Manipal University (MU) central animal
facility. The rats were housed in plastic cages of
size 36 cm 23 cm 21 cm (three rats in each cage)
inside a temperature- and humidity-controlled environment
with free access to food and water ad libitum,
with a 12 h light and 12 h dark cycle. All the experiments
were carried out with prior approval from the
institutional animal ethics committee. Care was taken
to handle the rats in a humane manner, and all
precautions were taken to use the minimum number
of animals required to generate significant data.
Experimental design
Animals were divided into two groups: group I
(n = 12), normal control; and group II (n = 12)
were exposed to RF-EMR by giving 50 missed calls
(within 1 hour) per day for 4 weeks, keeping a GSM
(0.9 GHz/1.8 GHz) mobile phone in vibratory mode
(no ring tone) in the cage (9). Each missed call was of
the duration of 45 seconds. Animals were free to move
in the cage. The phone was kept in a small woodbottomed
cage sized 12 cm 7 cm 7 cm. The
bamboo wire mesh on top of the wood bottom cage
prevented the animals from contact with the phone.
Twenty-four hours after the last exposure, six randomly
picked animals from both groups were tested
for passive avoidance behaviour using passive avoidance
apparatus. This test was conducted between
4.00 p.m. and 6.00 p.m. The remaining animals
from both groups were sacrificed to study the histological
changes in the hippocampus. Statistical analysis
was done by using Student’s t test. P-value < 0.05
was considered as significant.
Passive avoidance apparatus
The apparatus has two compartments, a rectangular
larger compartment with a 50 cm 50 cm grid floor
and wooden walls of 35 cm height. It has a roof, which
can be opened or closed. One of the walls has a
6 cm 6 cm opening connecting the larger compartment
to a dark smaller compartment. The smaller
compartment has a 15 cm 15 cm electrifiable grid
connected to a constant current stimulator, wooden
walls of 15 cm height, and a ceiling that can be
opened or closed. The connection between the two
compartments can be closed with a sliding door
made of Plexiglas. The larger compartment was
illuminated with a 100-W bulb placed 150 cm above
the centre.
Passive avoidance test
Passive avoidance test was conducted by the method
of Bures et al. (10), with modifications. The experiment
had three parts: 1) an exploration test, 2) an
aversive stimulation and learning test, and 3) a
retention test. The exploration test was conducted
in three trials. During this, each rat was kept in the
centre of the larger compartment facing away from
the entrance to the dark compartment. The door
between the two compartments was kept open. The
rat was allowed to explore the apparatus (both larger
and smaller compartments) for 3 minutes. In each
trial, the total time taken by the animal to enter the
dark compartment was noted using a stop-watch. At
the end of the trial, the rat was replaced in the home
cage, where it remained during an inter-trial interval
of 5 minutes. After the last exploration trial, the rat
was again kept in the larger compartment as in the
trial sessions. When the animal entered the smaller
compartment, the sliding door between the two
compartments of the apparatus was closed and three
strong foot shocks (50 Hz, 1.5 mA, and 1 s duration)
were given at 5-second intervals. The ceiling was
then opened and the rat was then returned to its
home cage. The retention test was carried out after
24 and 48 hours. The rat was kept in the centre of the
larger compartment facing away from the entrance
to the smaller compartment for a maximum period
of 3 minutes. The sliding door was kept open during
this period. The latency time required for the
animal to enter the dark compartment was recorded.
The latency time was recorded as 3 minutes for
those animals that did not enter the dark compartment
within 3 minutes. Absence of entry into the
dark compartment indicated positive memory
retention.
92 S. N. Narayanan et al.
Hematoxylin and eosin (H&E) staining
All histological procedures were uniform for control
and test group animals. The rats were sacrificed by
cervical dislocation under ether anaesthesia, and the
brain was exposed by cutting the skull along the midline.
The whole brain was carefully dissected out and
fixed in 10% buffered formalin (with pH 7.4) for 24 h.
It was then dehydrated in ethanol, defatted in xylene,
and embedded in paraffin. Care was taken to ensure
that all brains were oriented in the same direction
during embedding to minimize differences in the
angles at which the brains were sectioned. A single
investigator processed all brains to maintain consistency
in histological analysis. Sections were cut on a
rotary microtome (Leica RM2155, Germany) at
5-micron thickness and stained with hematoxylin
and eosin (H&E) according to standard procedure.
The hippocampal CA3 region was studied under a
light microscope. To avoid observer’s bias, an independent
person coded the slides before subjecting
them to morphological evaluations.
Results
Passive avoidance test
In the exploration trials, the entrance latency to the
dark compartment was decreased in both the groups
from first to third trial, but there was a significant
difference in the entrance latency time of the groups in
the second and third trials. The RF-EMR-exposed
animals took more time to enter the dark compartment
during the second and third exploration trials
(Figure 1).
During the memory retention test, the entrance
latency to the dark compartment was significantly
less for mobile phone-exposed rats when compared
with the control group. The latency was approximately
four times less in the mobile phone-exposed
animals tested 24 hrs after the shock trial (Figure 2A),
and the latency was approximately three times less in
the mobile phone-exposed rats tested 48 hours after
the shock trial (Figure 2B).
Hippocampal morphology
In comparison to the control animals, marked morphological
changes were detected in the CA3 region of
the hippocampus of the RF-EMR-exposed rats. The
hippocampus of RF-EMR-exposed rats showed
shrunken, darkly stained neurons (Figure 3B). No
such changes were observed in the control rats
(Figure 3A).
Discussion
Passive avoidance tests or conditioned avoidance tests
have been used in several studies to assess memory or
retention and also retrieval after or during other
treatments (11–13). Generally rats avoid intense illumination
and prefer dim illumination. When placed
in a brightly illuminated space connected with a dark
enclosure, they rapidly enter the dark compartment
and remain there. After an aversive consequence (foot
shock) in the dark compartment, the animals modify
their behaviour by inhibiting the innate activities or
learned habits (staying in the dark) and remain in the
bright compartment (10). So, in this task the animals
learn to avoid a noxious event by suppressing a
particular behaviour (14).
In the current study, the mobile phone exposure
significantly affected the passive avoidance behaviour
in rats. In other words, the memory retention and the
retrieval were significantly affected in mobile phone
RF-EMR-exposed rats. In comparison to the control
group, mobile phone-exposed animals showed
shorter latency to enter into the dark compartment
in the memory retention test (24 h and 48 h after the
aversive stimulus). This showed that the animals, after
being exposed to aversive stimulation (foot shock) in
the passive avoidance task, did not remember this task
to some extent on the following day, and this clearly
indicates the impairment of the memory. In mobile
phone-exposed animals the associative memory which
had built up through repetition over many trials and
expressed primarily in the performance of tasks
30
20
10
0
Trial 1 Trial 2
Control Exposed
Entrance latency to the dark
compartment (sec)
Trial 3
*
*
Figure 1. Time taken by the animals to enter the dark compartment
of the passive avoidance apparatus during the exploration trials
of passive avoidance test. The entrance latency to the dark compartment
was decreased in both the groups from first to third trial,
but there was a significant difference in the entrance latency time of
the groups in the second and third trials. The radio-frequency
electromagnetic radiation (RF-EMR)-exposed animals took
more time to enter the dark compartment during the exploration
trials. *P < 0.05.
Effect of RF-EMR on Wistar rats 93
was affected. This change in the behaviour of animals
(the shorter latency to enter the dark compartment) in
the passive avoidance task could be due to the altered
functioning of both hippocampal and amygdaloidal
neurons due to the damage caused by the RF-EMR
emitted from the mobile phone. A number of clinical
and experimental studies have shown the role of
hippocampal formation and related structures in
the medial temporal lobe in learning and memory
(15,16). In rats, bilateral lesion of the specific areas of
the hippocampus (CA1 and CA3) produced greater
impairments in the performance of passive avoidance
task (17). Bilateral hippocampal lesions in chicks
caused decreased retention of the avoidance response
(18). These studies suggest the involvement of the
hippocampal system in associative learning processes
and in memory.
In our current study, the hematoxylin and eosin
staining of the hippocampal region clearly showed
interspersed, deeply stained, shrunken cells, which
clearly indicates the degenerative changes in these
areas. The exact mechanism responsible for this
degeneration has to be investigated; perhaps the
mechanism is through reactive oxygen species. Earlier
A. B.
Figure 3. Representative photomicrograph of sections of hippocampal CA3 region of the brain from both control and radio-frequency
electromagnetic radiation (RF-EMR)-exposed rat stained with hematoxylin and eosin. A: Control animal; row of normal nerve cells in a section
of the pyramidal cell band of the hippocampus CA3 region is seen. B: Mobile phone RF-EMR-exposed rat; among the normal nerve cells, dark
(deeply stained) and shrunken nerve cells are seen.
A. 40 B.
30
20
10
0
Control Exposed
Entrance latency to the dark
compartment (sec)
*
Control
Entrance latency to the dark
compartment (sec)
Exposed
30 *
10
0
Figure 2. Effect of radio-frequency electromagnetic radiation (RF-EMR) on latency to enter the dark compartment 24 hours (A) and 48 hours
(B) after the shock trial. Rats exposed to the mobile phone took significantly less time to enter the dark compartment in the memory retention
test. Results are expressed as mean ± SEM. *P < 0.05.
94 S. N. Narayanan et al.
reports have stated that mobile phones caused oxidative
damage biochemically by increasing the levels of
Malondialdehyde (MDA), carbonyl groups, Xanthine
oxidase (XO) activity, and decreasing CAT activity;
and that treatment with melatonin significantly prevented
oxidative damage in the brain (19). The studies
on guinea-pigs have shown increases in MDA,
vitamins A, D3 (3), and E levels, increased CAT
enzyme activity, and decreased Glutathione (GSH)
level in the blood of Electromagnetic field (EMF)-
exposed guinea-pigs (20). The rats, when exposed to
900 MHz electromagnetic radiation from a mobile
phone for 7 days (1 h/day) showed 1) increase in
malondialdehyde and nitric oxide levels in brain tissue,
2) decrease in brain superoxide dismutase and
glutathione peroxidase activities, and 3) increase in
brain xanthine oxidase and adenosine deaminase
activities. Ginkgo biloba significantly prevented these
changes in the brain (21). Exposure of adult Sprague-
Dawley rats to regular cell phones resulted in mRNA
up-regulation of several injury-associated proteins,
such as calcium ATPase, neural cell adhesion molecule,
neural growth factor, and vascular endothelial
growth factor (22). The possible role of programmed
cell death in the brain could also not to be excluded.
Short-term exposure to cell phone radio-frequency
emissions (1900 MHz) can up-regulate elements of
apoptotic pathways in cells derived from the brain,
and neurons appear to be more sensitive to this effect
than are astrocytes (23). The primary neuronal cultures
of rats exposed to a continuous wave (CW) 900 MHz
Radiofrequency fields (RF) for 24 h induced apoptosis
through a caspase-independent pathway that involves
Apoptosis inducing factor (AIF) (24).
Both neurons and glia interact dynamically to
enable information processing and behaviour
(25,26). The poor performance of rats in the behavioural
tests could also be due to the damaging effect
of microwaves on glial cells, which in turn alters the
neuronal activity in the rat hippocampus and amygdala.
Acute exposure to GSM 900 MHz electromagnetic
fields (a single GSM exposure = 15 min)
induced glial reactivity and biochemical modifications
in the rat brain (27). Chronic exposure to GSM
900 MHz microwaves induced persistent astroglia
activation in the rat brain, which is the sign of a
potential gliosis (28). Reports also suggest that both
amygdala and hippocampus act synergistically to form
long-term memories of significantly emotional events,
and these brain structures are activated following an
emotional event and cross-talk with each other in the
process of consolidation (29). In order to prove the
involvement of various pathways (Reactive Oxygen
Species (ROS), apoptosis, or glial reactivation, or a
combination of all three) in the alteration of rat
behaviour and hippocampal morphology after
mobile phone RF-EMR exposure, further studies
are warranted.
Conclusion
The health effects of commonly encountered radiofrequency
electromagnetic radiations (RF-EMR) from
mobile phone exposures do exist. The evidence from
this study points to the quite substantial hazard of
RF-EMR from the mobile phone on passive avoidance
behaviour and hippocampal morphology in rats.
Declaration of interest: The authors report no
conflicts of interest. The authors alone are responsible
for the content and writing of this paper.
References
1. Finnie JW, Blumbergs PC, Manavis J, Utteridge TD,
Gebski V, Davies RA, et al. Effect of long-term mobile
communication microwave exposure on vascular permeability
in mouse brain. Pathology. 2004;36:96–7.
2. Aalto S, Haarala C, Brück A, Sipilä H, Hämäläinen H,
Rinne JO. Mobile phone affects cerebral blood flow in
humans. J Cereb Blood Flow Metab. 2006;26:885–90.
3. Kolesnyk IuM, Zhulins’ky ı VO, Abramov AV,
Kalinichenko MA. Effect of mobile phone electromagnetic
emission on characteristics of cerebral blood circulation and
neurohumoral regulation in humans. Fiziol Zh. 2008;54:90–3.
4. Finnie JW, Blumbergs PC, Cai Z, Manavis J, Kuchel TR.
Effect of mobile telephony on blood-brain barrier permeability
in the fetal mouse brain. Pathology. 2006;38:63–5.
5. Irmak MK, Fadillio glu E, Güleç M, Erdo gan H, Ya gmurca M,
Akyol O. Effects of electromagnetic radiation from a cellular
telephone on the oxidant and antioxidant levels in rats. Cell
Biochem Funct. 2002;20:279–83.
6. Tamasidze AG, Nikolaishvili MI. Effect of high-frequency
EMF on public health and its neuro-chemical investigations.
Georgian Med News. 2007:58–60.
7. Salford LG, Brun AE, Eberhardt JL, Malmgren L, Bertil R.
Nerve cell damage in mammalian brain after exposure to
microwaves from GSM mobile phones. Environ Health
Perspect. 2003;111:881–3.
8. Lai H, Singh NP. Single and double-stranded DNA breaks in
rat brain cells after acute exposure to radiofrequency electromagnetic
radiation. Int J Radiat Biol. 1996;69:513–21.
9. Narayanan SN, Kumar RS, Potu BK, Nayak S, Mailankot M.
Spatial memory performance of Wistar rats exposed to mobile
phone. Clinics. 2009;64:231–4.
10. Bures J, Buresova O, Huston JP. Techniques and basic
experiments for the study of brain and behavior. 2nd revised
and enlarged ed. Amsterdam, New York: Elsevier Science
Publishers; 1983. p. 148.
11. Bartus RT, Dean RL, Goas JA, Lippa AS. Age-related
changes in passive avoidance retention: modulation with
dietary choline. Science. 1980;209:301–3.
12. Glick SD, Crane AM, Barker LA, Mittag TW. Effect of
N-hydroxyl-pyrrolidinum methiodide, a choline analogue,
on passive avoidance behaviour in mice. Neuropharmacology.
1975;14:561–4.
Effect of RF-EMR on Wistar rats 95
13. Kopf SR, Buchholzer ML, Hilgert M, Löffelholz K, Klein J.
Glucose plus choline improve passive avoidance behaviour
and increase hippocampal acetylcholine release in mice.
Neuroscience. 2001;103:365–71.
14. Patterson CM, Kosson DS, Newman JP. Reaction to punishment,
reflectivity, and passive avoidance learning in extraverts.
J Pers Soc Psychol. 1987;52:565–75.
15. Liu X, Yang D, Meng Z. The effects of SO2 on electric activity
learning and memory of rat hippocampal neurons. Wei Sheng
Yan Jiu. 2008;37:660–3.
16. Zola-Morgan SM, Squire LR. The primate hippocampal
formation: evidence for a time-limited role in memory storage.
Science. 1990;250:288–90. [My paper].
17. Stubley-Weatherly L, Harding JW, Wright JW. Effects of
discrete kainic acid-induced hippocampal lesions on spatial
and contextual learning and memory in rats. Brain Res.
1996;716:29–38.
18. Sandi C, Rose SP, Patterson TA. Unilateral hippocampal
lesions prevent recall of a passive avoidance task in day-old
chicks. Neurosci Lett. 1992;141:255–8.
19. Sokolovic D, Djindjic B, Nikolic J, Bjelakovic G,
Pavlovic D, Kocic G, et al. Melatonin reduces oxidative stress
induced by chronic exposure of microwave radiation from
mobile phones in rat brain. J Radiat Res (Tokyo). 2008;
49:579–86.
20. Meral I, Mert H, Mert N, Deger Y, Yoruk I, Yetkin A, et al.
Effects of 900-MHz electromagnetic field emitted from cellular
phone on brain oxidative stress and some vitamin levels
of guinea pigs. Brain Res. 2007;1169:120–4.
21. Ilhan A, Gurel A, Armutcu F, Kamisli S, Iraz M, Akyol O,
et al. Ginkgo biloba prevents mobile phone-induced oxidative
stress in rat brain. Clin Chim Acta. 2003;111:881–3.
22. Yan JG, Agresti M, Zhang LL, Yan Y, Matloub HS.
Upregulation of specific mRNA levels in rat brain after cell
phone exposure. Electromagn Biol Med. 2008;27:147–54.
23. Zhao TY, Zou SP, Knapp PE. Exposure to cell phone
radiation up-regulates apoptosis genes in primary cultures
of neurons and astrocytes. Neurosci Lett. 2007;412:34–8.
24. Joubert V, Bouthoumieu S, Leveque P, Yardin C. Apoptosis is
induced by radiofrequency fields through the caspaseindependent
mitochondrial pathway in cortical neurons.
Radiat Res. 2008;169:38–45.
25. Laming PR, Kimelberg H, Robinson S, Salm A, Hawrylak N,
Müller C, et al. Neuronal-glial interactions and behaviour.
Neurosci Biobehav Rev. 2000;24:295–340.
26. Laming PR. Do glia contribute to behaviour? A neuromodulatory
review. Comp Biochem Physiol A. 1989;
94:555–68.
27. Brillaud E, Piotrowski A, de Seze R. Effect of an acute
900MHz GSM exposure on glia in the rat brain: a timedependent
study. Toxicology. 2007;238:23–33.
28. Ammari M, Brillaud E, Gamez C, Lecomte A, Sakly M,
Abdelmelek H, et al. Effect of a chronic GSM 900 MHz
exposure on glia in the rat brain. Biomed Pharmacother.
2008;62:273–81.
29. Richter-Levin G, Akirav I. Amygdala-hippocampus dynamic
interaction in relation to memory. Mol Neurobiol. 2000;
22:11–20.
96 S. N. Narayanan et al.