Evaluation of passive avoidance learning and spatial memory in rats exposed to low levels of lead during specific periods of early brain development

Abstract

Widespread use of heavy metal lead (Pb) for various commercial purposes has resulted in the environmental contamination caused by this metal. The studies have shown a definite relationship between low level lead exposure during early brain development and deficit in children's cognitive functions. This study investigated the passive avoidance learning and spatial learning in male rat pups exposed to lead through their mothers during specific periods of early brain development.
Experimental male rats were divided into 5 groups: i) the normal control group (NC) (N = 12) consisted of rat offspring born to mothers who were given normal drinking water throughout gestation and lactation, ii) the pre-gestation lead exposed group (PG) (N = 12) consisted of rat offspring, mothers of these rats had been exposed to 0.2% lead acetate in the drinking water for 1 month before conception, iii) the gestation lead exposed group (G) (N = 12) contained rat offspring born to mothers who had been exposed to 0.2% lead acetate in the drinking water throughout gestation, iv) the lactation lead exposed group (L) (N = 12) had rat offspring, mothers of these rats exposed to 0.2% lead acetate in the drinking water throughout lactation and v) the gestation and lactation lead exposed group (GL) (N = 12) contained rat offspring, mothers of these rats were exposed to 0.2% lead acetate throughout gestation and lactation.
The study found deficit in passive avoidance learning in the G, L and GL groups of rats. Impairment in spatial learning was found in the PG, G, L and GL groups of rats. Interestingly, the study found that gestation period only and lactation period only lead exposure was sufficient to cause deficit in learning and memory in rats. The extent of memory impairment in the L group of rats was comparable with the GL group of rats.
So it can be said that postnatal period of brain development is more sensitive to neurotoxicity compared to prenatal exposure.
This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.

Full text

533
ORIGINAL PAPER
International Journal of Occupational Medicine and Environmental Health 2015;28(3):533 – 544
http://dx.doi.org/10.13075/ijomeh.1896.00283
EVALUATION OF PASSIVE AVOIDANCE LEARNING
AND SPATIAL MEMORY IN RATS EXPOSED
TO LOW LEVELS OF LEAD DURING SPECIFIC
PERIODS OF EARLY BRAIN DEVELOPMENT
RAJASHEKAR RAO BARKUR1 and LAXMINARAYANA K. BAIRY2
1 Manipal University, Melaka Manipal Medical College, Manipal, India
Department of Biochemistry
2 Manipal University, Kasturba Medical College, Manipal, India
Department of Pharmacology
Abstract
Objectives: Widespread use of heavy metal lead (Pb) for various commercial purposes has resulted in the environmen-
tal contamination caused by this metal. The studies have shown a denite relationship between low level lead exposure
during early brain development and decit in children’s cognitive functions. This study investigated the passive avoid-
ance learning and spatial learning in male rat pups exposed to lead through their mothers during specic periods of early
brain development. Material and Methods: Experimental male rats were divided into 5 groups: i) the normal control
group (NC) (N = 12) consisted of rat offspring born to mothers who were given normal drinking water throughout gesta-
tion and lactation, ii) the pre-gestation lead exposed group (PG) (N = 12) consisted of rat offspring, mothers of these rats
had been exposed to 0.2% lead acetate in the drinking water for 1 month before conception, iii) the gestation lead exposed
group (G) (N = 12) contained rat offspring born to mothers who had been exposed to 0.2% lead acetate in the drinking
water throughout gestation, iv) the lactation lead exposed group (L) (N = 12) had rat offspring, mothers of these rats
exposed to 0.2% lead acetate in the drinking water throughout lactation and v) the gestation and lactation lead exposed
group (GL) (N = 12) contained rat offspring, mothers of these rats were exposed to 0.2% lead acetate throughout gestation
and lactation. Results: The study found decit in passive avoidance learning in the G, L and GL groups of rats. Impairment
in spatial learning was found in the PG, G, L and GL groups of rats. Interestingly, the study found that gestation period only
and lactation period only lead exposure was sufcient to cause decit in learning and memory in rats. The extent of memory
impairment in the L group of rats was comparable with the GL group of rats. Conclusions: So it can be said that postnatal
period of brain development is more sensitive to neurotoxicity compared to prenatal exposure.
Key words:
Rats, Developing brain, Blood lead, Hippocampus, Passive avoidance learning, Morris water maze
Received: April 3, 2014. Accepted: November 4, 2014.
Corresponding author: L.K. Bairy, Kasturba Medical College, Manipal University, Madav nagar, Manipal-576104, Karnataka, India (e-mail: kl.bairy@manipal.edu).
INTRODUCTION
Lead (Pb) has no biological role and is toxic to bio-
logical system. Lead has been widely used for vari-
ous commercial and industrial purposes because of its
economic value and easy availability. Lead is used in
manufacturing of lead-acid batteries, ceramic glaze,
plumbing materials, ammunition, wire sheathing, radia-
tion shielding material, herbal medicine, pigments and
paints [1–3]. In developing countries, environmental
contamination caused by heavy metals is on the increase
Nofer Institute of Occupational Medicine, Łódź, Poland

ORIGINAL PAPER R.R. BARKUR AND L.K. BAIRY
IJOMEH 2015;28(3)
534
their F1 offspring. Rats were housed in polypropylene
cages (37×21.5×14 cm) with a wire mesh lid and pad-
dy husk as bedding material. Animals were maintained
in 12L:12D cycle, in an air-conditioned room (22°C) and
controlled humidity in the central animal house facility. All
rats were fed with rat pellets (Amurut feed supplies, Puna,
India) and water ad libitum. The institutional animal ethical
committee’s (No. IAEC/KMC/07/2007-2008) approval was
obtained for the study. Animals were maintained and treat-
ed according to the guidelines recommended by the Com-
mittee for the Purpose of Control and Supervision of Ex-
periments on Animals, the Government of India.
Experimental design
Until the pregnancy in adult female rats, vaginal smears had
been collected every day and pro-estrous females had been
placed in separate cages along with male rats in the ratio 2:1
per cage. In the following morning, vaginal smear test was per-
formed. The presence of spermatozoa was taken as an index
of mating and pregnancy. This day was considered as GD1.
Pregnant females were individually housed in maternity cages.
The administration of lead (Pb) to the animals was per-
formed according to the previously published protocol
which had produced a blood lead levels 25–35 μg/dl in
adult rats maintained on lead [19,20]. This dosage was
selected as the blood lead levels 25–35 μg/dl seen in seg-
ments of population leaving in the area of environmental
lead contamination throughout the world (Toscano and
Guilarte, 2005) [21]. Lead acetate solution (0.2%) was
prepared in tap water and 0.5 ml/l glacial acetic acid was
added to prevent any precipitation of lead acetate.
The experimental pregnant rats were divided in-
to 5 groups (N = 30):
– normal control (NC) (N = 6),
– gestation lead exposed (G) (N = 6),
– lactation lead exposed (L) (N = 6),
– gestation and lactation lead exposed (GL) (N = 6),
– pre-gestation lead exposed (PG) (N = 6).
due to rapid economic development and limited regula-
tory infrastructure [4,5].
Epidemiological data has proven a denite relationship be-
tween low level lead exposure during early brain develop-
ment and impairment in children’s cognitive functions [6,7].
Studies have suggested that childhood lead exposure affects
various cognitive domains such as attention, executive func-
tion, visual-motor integration, social behavior, ne-motor
coordination and balance [8,9]. In children, the most widely
used chelator for lead toxicity is succimer [10]. However,
lead deposited in the brain cannot be removed by chemi-
cal chelating agents and reversal of the neurological de-
cits that are associated with elevated blood lead levels does
not take place by means of chelation therapy [11].
Crucial period of brain development in humans begins in
the last trimester of uterine life and continues till 2 years of
age. However, in rats, this phase of brain development ends
by the 4th week of life [12].
Hippocampal formation of the brain is critical for memory
storage, declarative memory and spatial navigation [13–15].
In humans, monkeys and rodents, the damage of hippocam-
pus negatively affects the performance in various learning
and memory tasks [16]. Rats with hippocampal lesions have
showed impaired spatial learning in the Morris water maze
test [14,17]. Kuhlmann et al. [18] found that rat pups exposed
to lead during gestation and lactation period had signicant
impairment in water maze performance, but not rats exposed
to lead after weaning. However, it would be interesting to nd
out whether only pre-gestation, only gestation, only lactation
period of lead exposure during early brain development can
cause learning and memory impairments. So, this study was
taken up to evaluate and compare the learning and memory
decits, if any, in rat pups exposed to low levels of lead during
specic periods of early brain development.
MATERIAL AND METHODS
The animals used in this study were adult male (200–
250 g) and female Wistar albino rats (180–210 g) and

EVALUATION OF MEMORY DEFICIT IN RATS EXPOSED TO LEAD ORIGINAL PAPER
IJOMEH 2015;28(3) 535
To meta-exchange reagent (2.9 ml), 100 μl of blood was
added using a micro-pipette and left undisturbed for 24 h.
The supernatant of the above solution mixture was used
for estimation of blood lead by anodic stripping voltam-
metry using ESA-3010B lead analyzer [22].
Behavioral experiments
The behavioral tests were performed from the postnatal
day 26 to 36. Memory retention was assessed by means of the
passive avoidance test and spatial memory was assessed using
the Morris water maze test. Before starting the behavioral
studies, the animals had been handled by the experimenter
so that they were free from the experimenter induced anxi-
ety. This was done by holding and stroking the experimental
animals by hand for the duration of 2–3 min each per animal
in 2 sessions with time intervals of 10 min.
Passive avoidance test
To test the memory retention, rats were subjected to the
passive avoidance learning test on the postnatal day 26 [23].
The test determines the ability of a rat to remember a foot
shock delivered 24/48 h prior to the memory retention test.
The passive avoidance apparatus consists of a wooden
box (50×50×35 cm) with a larger, brightly illuminated
compartment and a smaller (15×15×15 cm), dark compart-
ment with grid oor, which was attached to a shock source.
At the beginning of the experiment, a rat was placed in
the illuminated larger compartment for exploration.
The door between the 2 compartments was kept open dur-
ing this phase of the experimental period. The rat was al-
lowed to explore both of the compartments for 5 min. This
was followed by 3 test trials of 5 min each with an interval
of 30 min. At the end of 3rd test trial, as soon as the animal
had stepped into the dark compartment, the door between
the 2 compartments was closed and a single foot shock
was delivered through the grid oor (50 Hz, 1.5 mA,
for 1 s). The rat was held in the dark compartment
for an addi tional 10 s to allow the animal to form an
The rats in the gestation lead exposed group (G) were
given 0.2% lead acetate in drinking water throughout ges-
tation for 21 days. The rats in the lactation lead treated
group (L) were allowed to drink 0.2% lead acetate in drink-
ing water only after delivery for a period of 21 days (lac-
tation period). The gestation and lactation lead exposed
group (GL) of rats was administered 0.2% lead acetate in
drinking water throughout gestation and lactation periods,
which totaled to a period of 42 days. In the pre-gestation lead
exposed group, female rats were administered 0.2% lead ac-
etate in drinking water for the period of 30 days. These fe-
male rats of the pre-gestation lead exposed group were then
mated with the male rats and were maintained on normal
drinking water throughout the experimental period. The rats
in the normal control group (NC) were given normal water
to drink throughout gestation and lactation period.
Mothers of lead-exposed groups gave birth to healthy pups
without any physical deformities. There was no stillbirth.
On average, 3 to 4 male pups were born to each experimen-
tal pregnant rats out of which 2 male pups where randomly
selected for the experiment. So, the male rat pups (N = 12)
born to normal control rats and lead exposed rats (N = 12
in each group) were our experimental animals. On the post-
natal day 22, weaning of rat pups in the normal control and
lead exposed groups (GL, L, G and PG) was done and they
were housed in separate cages (4 rats per cage). From this
point onwards, rats of all groups were allowed normal water
for drinking for the rest of the experimental period. There
was no mortality of rat pups at weaning.
Birth weight and weight gain
Weights of male pups born to both the normal control and
lead exposed groups were measured on the day of birth
and on 7th, 14th, 21st postnatal days.
Estimation of blood lead
On the postnatal day 22, blood for lead estimation was
collected from orbital veins in a heparinized vacutainer.

ORIGINAL PAPER R.R. BARKUR AND L.K. BAIRY
IJOMEH 2015;28(3)
536
were analyzed using the video camera and Panlab Smart
Version 2.5 video tracking software, Barcelona, Spain.
The rats were trained in the water maze in 5 sessions
on 5 consecutive days; each session had 2 trials per ani-
mal with an interval of 2 h between the trials. During each
trial, different start points in the water tank were used.
In each trial, time taken by the rats to reach the hidden
platform was recorded and they were allowed to rest
for 10 s on the platform before removal from the tank.
The experimenter guided rats to the platform and if they
failed to nd the platform within 2 min, a maximum time
score of 2 min was assigned.
Twenty-four hours after the last learning session, rats
were subjected to the memory retention test. The memo-
ry retention session lasted 30 s. In this memory retention
test, the escape platform was removed from the target
quadrant and rat a was released in the quadrant opposite
to the target quadrant. Time taken to reach the target
quadrant, time spent in the target quadrant and distance
traveled in the target quadrant were measured. Greater
latency to reach the target quadrant and less time spent
in the target quadrant were indicative of spatial memory
impairment.
STATISTICAL ANALYSIS
Data was analyzed using the analysis of variance (ANO-
VA) followed by Bonferroni’s multiple comparison tests
as post hoc test (Graph pad prism 4 software). Values were
expressed in terms of mean±standard deviation (M±SD).
P < 0.05 were considered as signicant.
RESULT
Birth weight and weight gain
There was no signicant difference between birth
weight and weight gain in the lead exposed groups of
rat pups (GL, L, G and PG), when compared to the nor-
mal control group (NC) on the day of birth, 7th, 14th
and 21st postnatal day (Table 1).
association between the properties of the chamber and
the foot shock. It was then returned to its home cage. This
part of the experiment is called exploration and learning.
The memory retention test was done 24 h and 48 h af-
ter the foot shock. In the memory retention test, the rat
was placed in the bright chamber and time taken (the
step-through latency) to enter the dark compartment for
the 1st time was recorded using a stop-watch. A maximum
of 180 s was given for the rat to explore. Fraction of time
spent in the dark and bright compartments for each rat was
noted. Normal rats avoided entering the dark chamber,
where they received shock on the previous day, suppress-
ing their normal behavior of exploring the dark compart-
ment. Decrease in entry latency and decreased time spent
in bright compartment suggested poor memory retention.
Morris water maze test
To test the spatial memory, rats were subjected to the Mor-
ris water maze test from the postnatal day 30 till 36 [24].
The Morris water maze experiment consists of learning
session (for 5 days) and a memory retention test that took
place 24 h after the last learning session.
The water maze apparatus comprised of a white circu-
lar water tank of 1.8 m in diameter situated in a room il-
luminated by white uorescent lamps. Along the edge of
the water tanks, 4 points were marked and these divided
the pool into 4 equal quadrants. A square shaped escape
platform (4×4 inches size) was submerged (2 cm below
the water surface level) in 1 of the quadrant and this quad-
rant was called the target quadrant (TQ). The tank was
lled with 40 cm of tap water with the temperature of 26°C.
Before the experiment, powdered non-fat milk had been
added to make the water opaque. For facilitating the spatial
orientation in experimental animals, visual cues were placed
around the water tank. Positions of the cues were kept un-
changed throughout the period of experiment. A video
camera suspended from the ceiling above the tank was used
to record the experiment. Animal tracks in the water maze

EVALUATION OF MEMORY DEFICIT IN RATS EXPOSED TO LEAD ORIGINAL PAPER
IJOMEH 2015;28(3) 537
G: 70±23.74, L: 40.17±19.6, GL: 34.42±13.96) and GL,
L and G groups of rats spent less time (in s) in the bright
compartment as compared to the PG and NC groups of
rats (NC: 151.3±20.24, PG: 137.1±20.17, G: 87.08±28.96,
L: 70.25±27.77, GL: 68.58±36.52) (Figure 1).
Retention test after48h
Memory impairment in the GL, L, and G groups of rats
was evident as the entry latency data of this test was sim-
ilar to the latency (in s) to enter the dark compartment
Estimation of blood lead level on postnatal day 22
Blood lead level (mg/dl) was signicantly high in the gestation
and lactation lead exposed group (GL) of rats (32±1.97) fol-
lowed by the lactation lead exposed group (L) (26.65±4.08),
gestation lead exposed group (5.3±1.63), pre-gestation
lead exposed group (PG) (3.02±0.76), normal control
group (NC) (0.18±0.06) (Table 2).
Passive avoidance test
Exploration test
We did not nd any difference in the control and lead
exposed groups of rats (GL, L, G and PG) in the time
spent in the dark and bright chamber during exploration
in the passive avoidance test before receiving the foot
shock. The following data shows the time (in s) spent in
the dark compartment during exploration by different
groups of rats: NC: 252.92±26.67, PG: 245.83±21.20,
G: 251.25±19.55, L: 249.17±17.30, GL: 248.33±25.44.
Retention test after24h
The gestation and lactation lead exposed group (GL),
L and G groups of rats revealed impaired memory reten-
tion as they showed decreased latency (in s) to enter the
dark compartment (NC: 133.5±31.17, PG: 108.75±35.62,
Table 1. Body weight of rats of different groups
Study group
(N = 12)
Body weighta
(M±SD)
[g]
at birth postnatal
day 7
postnatal
day 14
postnatal
day 21
NC 5.50±0.83 11.03±0.85 18.96±1.86 25.17±1.75
PG 5.42±0.47 11.60±0.92 18.63±1.49 25.75±1.29
G 5.79±0.62 11.68±0.91 18.44±1.43 25.08±1.38
L 5.70±0.69 10.89±0.78 18.92±1.72 25.17±1.70
GL 5.56±0.54 11.63±0.63 18.40±1.16 25.83±1.40
a There is no signicance difference in body weight between the lead exposed groups as compared to the normal control group.
NC – normal control; PG – pregestation lead exposed; G – gestation lead exposed; L – lactation lead exposed; GL – gestation and lactation lead
exposed.
M – mean; SD – standard deviation.
Table 2. Blood lead (Pb) level in rats of different groups
(on the postnatal day 22)
Study group
(N = 10)
Pb
(M±SD)
[μg/dl]
NC 0.18±0.06
PG 3.02±0.76
G 5.30±1.63***
L 26.65±4.08***, ##, ###
GL 32.00±1.97***, #, ##, ###
Statistical signicance of results (one way ANOVA, Bonferroni’s test):
*** p < 0.001, when compared with NC; # p < 0.001, when compared
with PG; ## p < 0.001, when compared with G; ### p < 0.001, when
compared with L.
Other abbreviations as in Table 1.

ORIGINAL PAPER R.R. BARKUR AND L.K. BAIRY
IJOMEH 2015;28(3)
538
learnt to reach the escape platform much faster and their
escape latency decreased progressively from session to
session. However, lead treated groups of rats were taking
more time than normal control animals to reach the es-
cape platform (Figure 2).
Morris water maze retention test
Latency to enter target quadrants
There was a signicant increase in latency to enter
the target quadrants in the lead exposed group, name-
ly the GL, L, G and PG groups of rats as compared to
the NC (NC: 1.68±0.36, PG: 2.85±0.95, G: 4.83±1.92,
L: 5.47±1.96, GL: 8.55±2.64) (Figure 3).
Time spent in target quadrant
Spatial memory impairment in rats is also indicated by
the time spent in the target quadrant. The decreased
time spent in the target quadrant indicates spatial
after 24 h (NC: 82.92±37.75, PG: 81.83±31.06, G:
43.33±19.35, L: 25±12.82, GL: 22.25±15.41) and the GL,
L and G group of rat spent less time (in s) in the bright
compartment as compared to the PG and NC groups of
rats (NC: 107.5±30.26, PG: 88.33±33.39, G: 72.08±28.03,
L: 49.58±24.81, GL: 41.08±22.99) (Figure 1).
Morris water maze test
Learning sessions
During the learning sessions, latency to reach on to the es-
cape platform was measured in s. In the 1st session, rats in
all groups took equal amount of time to reach the escape
platform. In the 2nd session, rats in all groups were able
to reach the escape platform, much faster than during 1st
session. In the sessions no. 3, 4, and 5, rats in all groups
NC PG GLGL
0
30
60
90
120
150
180
24 48
NC PG GLGL
***
### ***
###
***
##
***
### ***
###
***
##
Latency [s]
***
###
***
###
***
###
***
##
***
#
*
Time [s]
0
30
60
90
120
150
180
a)
b)
24 48
Re
tention test [h]
Retention test [h]
Abbreviations as in Table 1.
Statistical signicance of results (one way ANOVA, Bonferroni’s test):
* p < 0.05, ** p < 0.01, *** p < 0.001, when compared with NC;
# p < 0.05, ## p < 0.01, ### p < 0.001, when compared with PG.
Each data represents mean ± standard deviation (M±SD) (N = 12
in each group).
Fig. 1. Passive avoidance performance of various rat groups
(on the postnatal day 27): a) latency to enter the dark
compartment, b) time spent in bright compartment
0
20
40
60
80
100
120
12345
Learning ession
s[day]
***
***
###
##
***
###
**
***
###
##
#
#
***
###
##
***
###
*
**
*
NC PG GLGL
Latency [s]
***
###
##
***
###
##
***
###
##
***
###
##
***
###
##
Abbreviations as in Table 1.
Statistical signicance of results (one way ANOVA, Bonferroni’s
test): * p < 0.05, ** p < 0.01, *** p < 0.001, when compared
with NC, ### p < 0.001, when compared with PG, ## p < 0.001,
when compared with G, # p < 0.01, when compared with L.
Each data represents M±SD (N = 6 in each group).
Fig. 2. Latency to escape on to the platform during learning
sessions in water maze of different rat groups (postnatal
day 30–34)

EVALUATION OF MEMORY DEFICIT IN RATS EXPOSED TO LEAD ORIGINAL PAPER
IJOMEH 2015;28(3) 539
memory impairment. The gestation and lactation lead-
exposed group (GL), L, G and PG groups of rats spent
signicantly less time in the target quadrant when com-
pared to the NC group (NC: 18.10±5.09, PG: 12.12±3.81,
G: 9.98±1.37, L: 7.67±1.21, GL: 5.20±3.15) (Figure 3).
Distance traveled in target quadrant
This data supplemented the results of time spent in the tar-
get quadrant data. The gestation and lactation lead ex-
posed group (GL), L, G and PG groups of rats showed sig-
nicantly shorter distance traveled in the target quadrant
when compared to the NC group (NC: 545.17±100.72,
PG: 374.11±12.40, G: 348.83±48.06, L: 291.00±57.20,
GL: 158.71±78.80) (Figure 3).
Video tracking of representative rats belonging to various
groups during memory retention test 24 h after the last
learning session in the water maze is given in the Figure 4.
DISCUSSION
There was no signicance difference between the birth
weight and weight gain (on 7th, 14th and 21st postnatal day)
in the lead exposed groups of rat pups (GL, L, G and PG),
when compared to the normal control group (NC). We
did not nd any still birth and mortality at weaning in the
lead exposed groups. So, it can be said that the low level
of environmentally relevant lead exposure dosage used
in the experiment did not cause any alteration in general
health of lead exposed animals.
Blood lead level was the highest in the GL group of rats
which had the longest time of lead exposure. This was fol-
lowed by the L, G, PG and NC group of rats, respectively.
The passive-avoidance test exploits the rodent’s prefer-
ence for darkness and assesses the long-term memory in
it [25]. In our study, we did not nd any difference in ex-
ploration behavior between the control and lead-exposed
groups of rats (GL, L, G and PG). During the retention
test (after 24 h and 48 h), the GL, L and G groups of
lead exposed rats showed lower latency to enter the dark
0
3
6
9
12
NC PG GLGL
***
###
*
*
Latency [s]
a) ##
0
6
12
18
24
NC PG GLGL
***
#
***
**
*
Time [s]
b)
0
100
200
300
400
500
600
700
c)
***
***
**
NC PG GLGL
Distance [cm]
***
##
####
Abbreviations as in Table 1.
Statistical signicance of results (one way ANOVA, Bonferroni’s test):
* p < 0.05,** p < 0.01, *** p < 0.001, when compared with NC,
# p < 0.05, ## p < 0.001, when compared with PG, ### p < 0.05,
#### p < 0.001, when compared with G.
Each data represents M±SD (N = 6 in each group).
Fig. 3. Performance in the water maze retention test of
various rat groups on the postnatal day 36: a) latency to
enter the target quadrant, b) time spent in target quadrant,
c) distance travelled in target quadrant

ORIGINAL PAPER R.R. BARKUR AND L.K. BAIRY
IJOMEH 2015;28(3)
540
pregnancy and lactation showed a decreased exploratory
behavior and impairment of learning and memory in the
shuttle box test. However, in this study, lead exposure only
during lactation (L group) and only during gestation pe-
riod (G group) caused memory decit in the passive avoid-
ance test. The extent of memory decit in the passive avoid-
ance test in the L group was comparable with that of the
GL group (lead exposure period of 42 days) even though
the period of exposure in L group was shorter (21 days).
The G group which had lead exposure for the period
of 21 days showed memory impairment in the passive avoid-
ance test which is comparatively shorter than the L group.
The impairment seen in the GL, L, and G group may
have been related to a possible inhibitory action of lead
on immature hippocampal neurons. Lead by interfering
with synapse formation might have contributed to de-
cits in passive avoidance memory formation. Hippocam-
pal dentate granule synapses gets stabilized after 6–8 h of
learning. Neural cell adhesion molecule (NCAM) plays
an important role in this process by inuencing dendritic
expansion and spine density [28]. The synthesis NCAM is
affected by lead [29].
The Morris water maze (MWM) test is used to test
the spatial memory in rodents to locate a hidden platform
in the pool of water using visual clues kept near the water
maze [14] and is closely associated with the number of gran-
ule cell neurogenesis in the dentate gyrus (DG) of hippo-
campus [30]. During the learning session (2nd to 5th) of the
water maze test, rats in all groups showed progressive de-
crease in escape latency from session to session. However,
lead treated groups of rats, namely the GL, L and G groups
were taking more time than normal control animals to reach
the escape platform. In the retention test 24 h after the learn-
ing session, there was a signicant increase in latency to enter
the target quadrants in the GL, L and G groups of rats. There
was also signicant reduction in time spent and the distance
traveled in the target quadrant in the GL, L, G and PG groups.
This conrms severe spatial memory impairment in lead
compartment than the control group and also they spent
less time in the bright compartment indicating lead in-
duced memory impairment.
De Oliveira et al. [26] found that rats exposed to low level
of lead from the gestation day 11 till the postnatal day 28
had signicantly reduced latency values in the step-through
inhibitory avoidance memory task. In a study conducted by
Moreira et al. [27], rat pups exposed to lead acetate during
PG, G, L and GL group of rats spent less time, and traveled a short
distance in the target quadrant during the water maze test indicating
spatial memory impairment.
B – beginning of the track; TQ – target quadrant; E – end of the track.
Other abbreviations as in Figure 1.
Fig. 4. Video tracking of representative rats belonging to
different groups during memory retention test 24 h after last
learning session in water maze

EVALUATION OF MEMORY DEFICIT IN RATS EXPOSED TO LEAD ORIGINAL PAPER
IJOMEH 2015;28(3) 541
signicant learning and memory retrieval decits in young
offspring both in the passive avoidance test and Morris
water maze test. Interestingly, in both of the above tests
the extent of memory impairment in the L group of rats
was comparable with the GL group of rats. However,
pre-gestation lead exposure had minimal ill effects on
memory impairment as compared to other lead exposed
groups. It can also be noted that, blood lead level on the
postnatal day 22 in the G group (5.30±1.63 μg/dl) were
almost simi lar to the PG group (3.02±0.76 μg/dl). Never-
theless, the G group showed signicant memory impair-
ment as compared to the PG group. This fact proves that
it was the period of lead exposure during brain develop-
ment which was responsible for extent of memory impair-
ment seen in the G group rather than blood lead level at
the time of learning and memory assessment. This fact
holds true also for the memory impairment seen in the L
and GL group of rats.
CONCLUSIONS
In conclusion, this study demonstrates that lactation pe-
riod brain development is a sensitive period as far as lead
exposure is concerned. The reasons for this may be nu-
merous. But, we hypothesize that development of dentate
gyrus of hippocampus, which occurs mainly from the post-
natal day 1 to the postnatal day 19 [43], gets affected as
a consequence of lead exposure in the lactation period.
The 2nd reason may consist in the fact that postnatal neu-
ronal differentiation and synaptogenesis might have been
affected by lead exposure from the postnatal day 1 to 21
in both the L and GL groups of rats. So, it can be said
that the postnatal period of brain development is more
sensitive to lead neurotoxicity when compared to that of
prenatal period.
ACKNOWLEDGMENTS
The authors thank the Manipal University, India for providing
the infrastructure facilities.
exposed groups especially in the GL, G and L groups which
may be due to the attention decit.
Previous studies have found that chronic lead exposure
in Wistar rats during gestation and lactation resuld in
decreased learning performances in the water maze
test [18,31] and cause damage to mitochondria, micro-
laments, and microtubules in hippocampal neurons [32].
Chang et al. [33] showed spatial learning decits in the
Morris water maze test, in rats exposed to 0.2% lead acetate
in the drinking water through their mothers from the ges-
tational day 15 to the postnatal day 21. Yang et al. [34] and
Wang et al. [35] concluded from their study that exposure to
lead during only gestational period and only lactation peri-
od is sufcient to cause spatial memory decits in the Mor-
ris water maze test in young adult offspring respectively.
In this study, it has been demonstrated that lead ex-
posure during only gestation period and only lactation pe-
riod produces signicant impairment in water maze test.
Cognitive decit in the L group was comparable to the
GL group even though the period of lead exposure was
much shorter (L group: 21 days vs. GL group: 42 days).
Exposure to lead during early brain development produces
hippocampal synaptic plasticity decits and affects long-
term potentiation (LTP) and spatial memory in young adult
rats [36,37]. The reason for this phenomenon has been at-
tributed to selective inhibition of N-methyl-D-aspartate
subtype of excitatory receptors (NMDAR) by lead [38,39].
Lead alters the expression of the NMDAR subunits result-
ing in the defective NMDAR structure affecting the hip-
pocampal development and maturation [35,36,40]. Lead is
believed to inuence the expression of hippocampal DNA
methyltransferases and methyl cytosine-binding proteins
which are involved in early brain development process [41].
Lead exposure from the gestation day 0 to the postnatal
day 21 in rats, enhances oxidative stress and alters the apop-
tosis process in developing hippocampal neurons [42].
This study has revealed that the gestation period (G group)
and only lactation period (L group) lead exposure causes

ORIGINAL PAPER R.R. BARKUR AND L.K. BAIRY
IJOMEH 2015;28(3)
542
to lead. N Engl J Med. 2001;344(19):1421–6, http://dx.doi.
org/10.1056/NEJM200105103441902.
12. Rice D, Barone S Jr. Critical periods of vulnerability for
the developing nervous system: Evidence from humans and
animal models. Environ Health Perspect. 2000;108 Sup-
pl 3:511–33.
13. O’Keefe J, Nadel L. The hippocampus as a cognitive map.
Oxford: Oxford University Press; 1978. p. 62–89.
14. Morris RGM, Garrud P, Rawlins JNP, O’Keefe J.
Place navigation impaired in rats with hippocampal le-
sions. Nature. 1982;297(5868):681–3, http://dx.doi.org/10.
1038/297681a0.
15. Fortin NJ, Agster KL, Eichenbaum HB. Critical role of
the hippocampus in memory for sequences of events. Nat
Neurosci. 2002;5(5):458–62, http://dx.doi.org/10.1038/nn834.
16. Eichenbaum H. The hippocampus and declarative memo-
ry: Cognitive mechanisms and neural codes. Behav Brain
Res. 2001;127(1–2):199–207, http://dx.doi.org/10.1016/S01
66-4328(01)00365-5.
17. Pearce JM, Roberts AD, Good M. Hippocampal lesions
disrupt navigation based on cognitive maps but not head-
ing vectors. Nature. 1998;396(6706):75–7, http://dx.doi.
org/10.1038/23941.
18. Kuhlmann AC, McGlothan JL, Guilarte TR. Developmen-
tal lead exposure causes spatial learning decits in adult
rats. Neurosci Lett. 1997;233(2–3):101–4, http://dx.doi.
org/10.1016/S0304-3940(97)00633-2.
19. Jaako-Movits K, Zharkovsky T, Romantchik O, Jurgen-
son M, Merisalu E, Heidmets LT, et al. Developmental
lead exposure impairs contextual fear conditioning and re-
duces adult hippocampal neurogenesis in the rat brain. Int
J Dev Neurosci. 2005;23(7):627–35, http://dx.doi.org/10.10
16/j.ijdevneu.2005.07.005.
20. Chen HH, Ma T, Paul IA, Spencer JL, Ho IK. Develop-
mental lead exposure and two-way active avoidance train-
ing alter the distribution of protein kinase C activity
in the rat hippocampus. Neurochem Res. 1997;22(9):
1119–25, http://dx.doi.org/10.1023/A:1027365202328.
REFERENCES
1. Falk H. International environmental health for the pe-
diatrician: Case study of lead poisoning. Pediatrics.
2003;112(1 Pt 2):259–64.
2. Agency for Toxic Substances and Disease Registry (ATSDR)
Toxicological prole for lead. Atlanta (GA): US Depart-
ment of Health and Human Services; 2007. p. 102–225.
3. Acharya S. Lead between the lines. Nat Chem. 2013;
5(10):894, http://dx.doi.org/10.1038/nchem.1761.
4. Yáñez L, Ortiz D, Calderón J, Batres L, Carrizales L, Me-
jía J, et al. Overview of human health and chemical mixtures:
Problems facing developing countries. Environ Health Per-
spect. 2002;110 Suppl 6:901–9.
5. Trasande L, Massey RI, DiGangi J, Geiser K, Ola-
nipekun AI, Gallagher L. How developing nations can
protect children from hazardous chemical exposures
while sustaining economic growth. Health Aff (Mill-
wood). 2011;30(12):2400–9, http://dx.doi.org/10.1377/hl
thaff.2010.1217.
6. Needleman HL. The current status of childhood low-level
lead toxicity. Neurotoxicology. 1993;14(2–3):161–6.
7. Markowitz M. Lead poisoning: A disease for the next mil-
lennium. Curr Probl Pediatr. 2000;30(3):62–70, http://dx.
doi.org/10.1067/mps.2000.104053.
8. Lanphear BP, Dietrich K, Auinger P, Cox C. Cognitive de-
cits associated with blood lead concentrations 10 microg/dL
in US children and adolescents. Public Health Rep. 2000;
115(6):521–9.
9. Bellinger DC. Effect modication in epidemiological
studies of low-level neurotoxicant exposures and health
outcomes. Neurotoxicol Teratol. 2000;22(1):133–40, http://
dx.doi.org/10.1016/S0892-0362(99)00053-7.
10. Warniment C, Tsang K, Galazka SS. Lead poisoning in chil-
dren. Am Fam Physician. 2010;81(6):751–7.
11. Rogan WJ, Dietrich KN, Ware JH, Dockery DW, Salgan-
ik M, Radcliffe J, et al. Treatment of Lead-Exposed Children
Trial Group. The effect of chelation therapy with succimer
on neuropsychological development in children exposed

EVALUATION OF MEMORY DEFICIT IN RATS EXPOSED TO LEAD ORIGINAL PAPER
IJOMEH 2015;28(3) 543
Proc Natl Acad Sci U S A. 2003;100(24):14385–90, http://
dx.doi.org/10.1073/pnas.2334169100.
31. Kahloula K, Slimani M, Dubois M, Bonnet J. D-cycloser-
ine enhances spatial learning performances of rats chroni-
cally exposed to lead during the developmental period.
Afr J Neurol Sci. 2009;28(1):67–77, http://dx.doi.org/10.43
14/ajns.v28i1.55141.
32. Xu J, Yan HC, Yang B, Tong LS, Zou YX, Tian Y. Effects
of lead exposure on hippocampal metabotropic glutamate re-
ceptor subtype 3 and 7 in developmental rats. J Negat Results
Biomed. 2009;8:5, http://dx.doi.org/10.1186/1477-5751-8-5.
33. Chang W, Chen J, Wei QY, Chen XM. Effects of Brn-3a
protein and RNA expression in rat brain following low-
level lead exposure during development on spatial learning
and memory. Toxicol Lett. 2006;164(1):63–70, http://dx.doi.
org/10.1016/j.toxlet.2005.11.011.
34. Yang Y, Ma Y, Ni L, Zhao S, Li L, Zhang J, et al. Lead
exposure through gestation-only caused long-term learn-
ing/memory decits in young adult offspring. Exp Neu-
rol. 2003;184(1):489–95, http://dx.doi.org/10.1016/S0014-
4886(03)00272-3.
35. Wang XM, Liu WJ, Zhang R, Zhou YK. Effects of exposure
to low-level lead on spatial learning and memory and the ex-
pression of mGluR1, NMDA receptor in different develop-
mental stages of rats. Toxicol Ind Health. 2013;29(8):686–96,
http://dx.doi.org/10.1177/0748233712436641.
36. Nihei MK, Desmond NL, McGlothan JL, Kuhlmann AC,
Guilarte TR. N-methyl-D-aspartate receptor subunit
changes are associated with lead- induced decits of
long-term potentiation and spatial learning. Neurosci-
ence. 2000;99(2):233–42, http://dx.doi.org/10.1016/S0306-
4522(00)00192-5.
37. Guilarte TR, Toscano CD, McGlothan JL, Weaver SA. En-
vironmental enrichment reverses cognitive and molecular
decits induced by developmental lead exposure. Ann Neu-
rol. 2003;53(1):50–6, http://dx.doi.org/10.1002/ana.10399.
38. Alkondon M, Costa AC, Radhakrishnan V, Aronstam RS,
Albuquerque EX. Selective blockade of NMDA-activated
21. Toscano CD, Guilarte TR. Lead neurotoxicity: From expo-
sure to molecular effects. Brain Res Rev. 2005;49(3):529–54,
http://dx.doi.org/10.1016/j.brainresrev.2005.02.004.
22. Kuruvilla A, Pillay VV, Venkatesh T, Adhikari P, Chakrapa-
ni M, Clark CS, et al. Portable lead analyzer to locate source
of lead. Indian J Pediatr. 2004;71(6):495–9, http://dx.doi.
org/10.1007/BF02724287.
23. Bureš J, Burešová O, Huston JP. Techniques and basic ex-
periments for the study of brain and behavior. 2nd ed. Am-
sterdam, New York: Elsevier; 1983. p. 95–7, 148–60.
24. D’Hooge R, de Deyn PP. Applications of the Morris wa-
ter maze in the study of learning and memory. Brain Res
Rev. 2001;36(1):60–90, http://dx.doi.org/10.1016/S0165-01
73(01)00067-4.
25. Dawson GR, Heyes CM, Iversen SD. Pharmacologi-
cal mechanisms and animal models of cognition. Behav
Pharmacol. 1992;3(4):285–97, http://dx.doi.org/10.1097/00
008877-199208000-00003.
26. De Oliveira FS, Viana MR, Antoniolli AR, Marchioro M.
Differential effects of lead and zinc on inhibitory avoidance
learning in mice. Braz J Med Biol Res. 2001;34(1):117–20,
http://dx.doi.org/10.1590/S0100-879X2001000100014.
27. Moreira EG, Vassilieff I, Vassilieff VS. Developmental
lead exposure: Behavioral alterations in the short and long
term. Neurotoxicol Teratol. 2001;23(5):489–95, http://dx.doi.
org/10.1016/S0892-0362(01)00159-3.
28. Doyle E, Nolan PM, Bell R, Regan CM. Intraventricular in-
fusions of anti-neural cell adhesion molecules in a discrete
posttraining period impair consolidation of a passive avoid-
ance response in the rat. J Neurochem. 1992;59(4):1570–3,
http://dx.doi.org/10.1111/j.1471-4159.1992.tb08477.x.
29. Cookman GR, King W, Regan CM. Chronic low-level lead
exposure impairs embryonic to adult conversion of the neu-
ral adhesion molecule. J Neurochem. 1987;49(2):399–403,
http://dx.doi.org/10.1111/j.1471-4159.1987.tb02879.x.
30. Drapeau E, Mayo W, Aurousseau C, Le Moal M, Piazza PV,
Abrous DN. Spatial memory performance of aged rats in
the water maze predicts level of hippocampal neurogenesis.

ORIGINAL PAPER R.R. BARKUR AND L.K. BAIRY
IJOMEH 2015;28(3)
544
41. Schneider JS, Kidd SK, Anderson DW. Inuence of
developmental lead exposure on expression of DNA
methyltransferases and methyl cytosine-binding pro-
teins in hippocampus. Toxicol Lett. 2013;217(1):75–81,
http://dx.doi.org/10.1016/j.toxlet.2012.12.004.
42. Lu X, Jin C, Yang J, Liu Q, Wu S, Li D, et al. Prenatal and
lactational lead exposure enhanced oxidative stress and
altered apoptosis status in offspring rats’ hippocampus.
Biol Trace Elem Res. 2013;151(1):75–84, http://dx.doi.org/
10.1007/s12011-012-9531-5.
43. Bayer SA, Altman J, Russo RJ, Zhang X. Timetables of neu-
rogenesis in the human brain based on experimentally deter-
mined patterns in the rat. Neurotoxicology. 1993;14(1):83–144.
channel currents may be implicated in learning decits
caused by lead. FEBS Lett. 1990;261(1):124–30, http://
dx.doi.org/10.1016/0014-5793(90)80652-Y.
39. Gavazzo P, Gazzoli A, Mazzolini M, Marchetti C. Lead
inhibition of NMDA channels in native and recombinant
receptors. Neuroreport. 2001;12(14):3121–5, http://dx.doi.
org/10.1097/00001756-200110080-00028.
40. Guilarte TR, McGlothan JL, Nihei MK. Hippocam-
pal expression of N-methyl-D-aspartate receptor (NM-
DAR1) subunit splice variant mRNA is altered by de-
velopmental exposure to Pb(2+). Brain Res Mol Brain
Res. 2000;76(2):299–305, http://dx.doi.org/10.1016/S0169-
328X(00)00010-3.
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