Biological Trace Element Research

https://doi.org/10.1007/s12011-018-1452-5

Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic

and Dopaminergic System in Brain of Developing Rats
Lalit P. Chandravanshi 1,2 & Richa Gupta 2 & Rajendra K. Shukla 3

Received: 10 May 2018 / Accepted: 18 July 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
Chronic exposure to arsenic via drinking water throughout the globe is assumed to cause a developmental neurotoxicity. Here, we
investigated the effect of perinatal arsenic exposure on the neurobehavioral and neurochemical changes in the corpus striatum,
frontal cortex, and hippocampus that is critically involved in motor and cognition functions. In continuation of previous studies,
this study demonstrates that perinatal exposures (GD6–PD21) to arsenic (2 or 4 mg/kg body weight, p.o.) cause hypo-activity in
arsenic-exposed rats on PD22. The hypo-activity was found to be linked with a decrease in the mRNA and protein expression of
the DA-D2 receptor. Further, a protein expression of tyrosine hydroxylase (TH), levels of dopamine, and its metabolites were also
significantly impaired in corpus striatum. The arsenic-exposed groups showed spatial learning and memory significantly below
the average in a dose-dependent manner for the controls. Here, we evaluated the declined expression of CHRM2 receptor gene
and protein expression of ChAT, PKCβ-1 in the frontal cortex and hippocampus, which are critically involved in cognition
functions including learning and memory. A trend of recovery was found in the cholinergic and dopaminergic system of the brain,
but changes remained persisted even after the withdrawal of arsenic exposure on PD45. Taken together, our results indicate that
perinatal arsenic exposure appears to be critical and vulnerable as the development of cholinergic and dopaminergic system
continues during this period.

Keywords Developmental neurotoxicity . Arsenic . DA-D2 receptor . Spatial memory . Locomotor activity etc.

Introduction Argentina, Mexico, and Colombia, which have high arsenic

Arsenic is one of the environmental neurotoxicant and ubiq-
uitous in nature due to its natural existence and anthropogenic
sources [49]. As per an estimate, over 200 million people have
been estimated to be chronically exposed to arsenic in drink-
ing water throughout the globe [44, 84]. Higher incidence of
cancer, arsenicosis, skin lesions, cardiovascular diseases, re-
productive problems, and psychological, neurological, and
mental health problems is reported in many countries like
India, Bangladesh, Japan, Argentina, Bolivia, Taiwan, Chile,
* Lalit P. Chandravanshi
chandravanshi04@gmail.com
1 levels of arsenic have been detected in the breast milk of
Division of Forensic Science, School of Basic and Applied Sciences,
Galgotias University, Greater Noida 201307, India
2 fects on infants [20]. These findings suggest that arsenic
Developmental Toxicology Division, CSIR–Indian Institute of
Toxicology Research, Post Box No. 80, MG Marg, Lucknow 226
001, India
3
Department of Biochemistry, All India Institute of Medical Sciences,
Bhopal, India
contamination in the drinking water [9, 14, 16, 26, 28, 33, 40].
Growing evidence for animal and human studies indicated
that arsenic might have deleterious effects on the central ner-
vous systems [53, 89]. Furthermore, concern over develop-
mental neurotoxicity of arsenic has been raised in the present
scenario [25, 79]. Arsenic exposure could produce develop-
mental abnormalities, including malformations, decreased
growth rate, mortality, neuronal tube defects [46, 60, 68],
and an array of behavioral changes through effects on the
developing brain directly since arsenic freely crosses the pla-
centa and blood–brain barrier (BBB) in humans and animals
[27, 31, 66, 67, 85]. Neural tube defects, spontaneous abor-
tion, stillbirth, and neonatal deaths were reported in arsenic-
exposed pregnant women who are consuming drinking water
contaminated with high arsenic levels [46, 48, 58, 59]. Low
Bangladeshi women and associated with adverse health ef-
may cause to impaired fetal and infant health.
Slow nerve conduction velocity, and sensory and motor
dysfunctions have also been reported in arsenic-exposed pop-
ulation [55, 76]. Many investigators suggest that arsenic

exposure to maternal and early life can affect the functioning
of the central nervous system [70, 74]. Arsenic can alter the
motor and cognitive functions by the dysfunctions of multiple
neurobiological processes including those of dopaminergic,
cholinergic, glutamatergic, and monoaminergic signaling
pathways [10, 63, 87]. Arsenic can delay and inhibit the pro-
cess of neurodevelopment of the central nervous system dur-
ing the fetal and early life of the human [61]. Several scientists
have investigated that arsenic could be modulating the dopa-
minergic system and associated behaviors. Dopaminergic
neurons are important to usual dopaminergic neurotransmis-
sion signaling and involved in modulating reward, motivation,
and motor activity functions by interacting with its receptors
[18, 69]. TH is a rate-limiting enzyme in the biosynthesis of
dopamine. Meanwhile, DA-D2 receptors in the striatum are
functionally involved in modulating the movement and cog-
nition [22]. Arsenic may significantly alter the levels of DA
and their metabolites in the brain and thus affects the neuro-
chemical and behavioral abnormalities [1, 7, 30, 35, 47, 62].
Muscarinic–cholinergic receptors play major roles in the
memory function, and evidence suggests a role in neurological
disorders like Alzheimer’s disease and schizophrenia [32, 50].
Acetylcholine coordinates the neuronal networks of the cho-
linergic modulation area of the brain. Synthesis and function
of acetylcholine are regulated by the choline acetyltransferase
(ChAT) and acetylcholinesterase (AChE) enzymes [80].
Cholinergic dysfunctions could be associated with the impair-
ment in motor coordination and learning, and could arise from
cholinergic dysfunctions. Reduced AChE and ChAT along
with the degeneration of cholinergic neurons have been re-
ported in arsenic-exposed rats and provide support for arsenic
in the etiology of Alzheimer’s disease [52]. PKC β-1 is a
molecule crucially involved in long-term potentiation and dif-
ferent forms of learning [54, 86]. PKC activation induces pro-
tein synthesis required for long-term memory. It appears intu-
itive that in some diseases, alterations in PKC β-1 cause or
enhance the risk of learning and memory impairments, an idea
that will need testing in arsenic-induced developmental neu-
rotoxicity. Further, deficits in cognition and motor function
following acute and chronic exposure to high dose of arsenic
have been reported on adult animals [63, 87]. Mechanisms
underlying the neurotoxic effects of arsenic are not well un-
derstood during a developing period of the brain. Most of the
researches on the neurobiological consequences of arsenic
exposure have focused largely on adult animals at high doses.
Generally, humans getting exposed to low level of arsenic
instead of high level is close to real-life exposure scenarios
and is of greatest concern for arsenic exposure. The nervous
system will respond to the neurotoxic insult depending on the
stage of brain development. Therefore, the timing of exposure
is very critical to the developmental neurotoxicity. Perinatal
period of rats is very crucial to the brain development as struc-
tural and functional maturation takes place during this period.
Chandravanshi et al.
The period GD6–PD21 is the developmental period in rats
during which maturation of different neurotransmitter sys-
tems, cholinergic, dopaminergic, and serotonergic receptors,
BBB, the setting of neurons, and synaptogenesis occurs [38].
However, the impact of arsenic exposure on the neurobehav-
ioral functions and its associated cholinergic and dopaminer-
gic system is not well understood during this perinatal period
of brain development. Based on the previous findings [11, 12],
this article generates a hypothesis by showing the need to
address the cholinergic and dopaminergic dysfunctions along
with neurobehavioral deficits in perinatally arsenic (2 or 4 mg/
kg/p.o.) exposed rats on PD22. This study also assessed the
transient or persistent effect of arsenic even after 23 days of
withdrawal of arsenic exposure.
Materials and Methods
Animal and Treatment
Perinatal exposure of rats to sodium arsenite: Adult female
rats of proven fertility were kept for mating with the males
in the ratio of 2:1. The day of appearance of the vaginal plug
was considered as day 1 (GD-1) of gestation and the pregnant
rats were separated into individual cages. Pregnant rats were
divided into three groups (six pregnant rats in each group) and
treated with sodium arsenite (SIGMA, USA) on day 6 of
gestation (GD-6) till postnatal day (PD) 21 as follows:
Group I—Pregnant rats were treated with normal saline
(p.o.) identically as in groups II and III and served as
controls.
Group II—Pregnant rats were treated with sodium arse-
nite (2 mg/kg body weight, p.o.) dissolved in distilled
water once daily from GD6 to PD21.
Group III—Pregnant rats were treated with sodium arse-
nite dissolved in distilled water (4 mg/kg body weight,
p.o.) once daily from GD6 to PD21.
All pregnant rats were allowed to deliver naturally and rear
their pups to weaning until PD21. All the litters were culled on
PD2; 8–10 pups with an equal allocation of males and females.
Cage-side observations were made to assess the adverse effects
of arsenic if any of the pups [39]. Development of physical
milestones and reflexes were assessed following the standard
protocol as described by Vorhees et al. [82]. Pup viability, av-
erage pup weight, and sex ratios were also observed from PD1
to PD21. In this experiment, pups were exposed to arsenic
through the mother from GD6 (gestational exposure) to PD21
(lactational exposure). On PD22, male rats were separated and
effect of arsenic on other behavioral endpoints studied on
PD22. The animals were sacrificed and the brain was taken
out quickly, washed in ice-cold saline, and dissected into
Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic and Dopaminergic System in Brain of Developing...
specific regions—frontal cortex, corpus striatum, and hippo-
campus following the standard procedure as described by
Glowinski and Iversen [23]. The dissected brain regions were
stored at − 80 °C for neurochemical assays. Unfortunately, we
did not evaluate the effect of arsenic on female pups. To explore
whether these changes produced by arsenic were transient and
persistent, another set of experiment was done on the same
conditions as mentioned above. Male pups were separated from
mothers on PD22 in each treatment group and maintained as
such till PD45. Neurobehavioral and neurochemical endpoints
were then assessed on PD45.
Behavioral Studies
Developmental Landmarks and Reflexes
The effects on the development of physical milestones and
reflexes were studied in rat pups perinatally exposed to arsenic
from GD6 to PD21. The test battery as described by Lazarini
et al. [39] was followed to assess the criteria of development
of physical milestones and reflexes.
Physical developmental milestones, viz., eye-opening, pin-
na detachment, incisor eruption, and growth of fur, were ob-
served each day, beginning on PD1.The following reflex pa-
rameters were assessed in the rats of control and arsenic-
exposed groups (2 or 4 mg/kg) as mixed sex: surface righting,
negative geotaxis, and cliff avoidance. Separate pups (ten)
were used for individual reflex parameters.
I. Surface righting reflex: The ability of rat pups to right
itself was determined on every alternate day from PD1
through PD7. Rat pups were placed on their back on
the clean body surface and the time required to turn
over to ventral surface in normal upright position re-
corded. A criterion of successful righting within 2 s
was used. The day all rat pups per litter reached the
criterion was recorded [71].
II. Negative geotaxis reflex: Negative geotaxis was evaluat-
ed using the procedure described by Farag et al. [21]. The
plane was made of wood and each test rat was given one
trial on PD5, PD10, and PD15. The trial was considered
to be a success when the pup turned 180° within the
allotted time of 60 s. The time required for rats to reorient
toward the top when placed in a head-down position on a
board inclined to 25°, and the latency to rotate 180° was
measured with the number of successful trials.
III. Cliff avoidance reflex: Cliff avoidance was evaluated on
PD4 and pups were placed on a wooden platform elevat-
ed 20 cm above a table top with the forepaws and nose
over the edge. The time required to return the body
1.5 cm from the Bcliff^ was recorded. A successful re-
sponse to rats within 30 s per trial was recorded [43].
Spontaneous Motor Activity
Spontaneous motor activity was evaluated by using auto-
mated Actimot system (TSE, Germany) following the
standard procedure as described by Chandravanshi et al.
[12]. A square-shaped frame was equipped with a high
number and density of infrared beams (32 × 32) in this
apparatus. The activity monitor operates on light beam
principle. Briefly, rats were acclimatized to half an hour
in behavioral room. The apparatus was cleaned and dried
with an ethanol solution (10%) before use every time.
Following this, each rat was individually placed in the
center of the cage (45 cm × 45 cm). As soon as the ani-
mals are placed in the cage, sensors become active and
counting device starts. Activity was monitored for 5 min
and parameters—total distance traveled, resting time, time
moving, and stereotypical time—were recorded simulta-
neously. Data collected by the system software were ana-
lyzed and the effect of different parameters assessed in
rats in the control and arsenic-exposed rats.
Grip Strength
Forelimb grip strength was assessed in the control and arsenic-
exposed rats by using a computerized grip strength meter
(TSE Germany) following the standard procedure as de-
scribed by Terry Jr et al. [73]. Holding the rat by the neck
and the base of the tail, the forelimbs were placed on the
tension bar. The rat was pulled back gently until it released
the bar. Reading was recorded automatically on the computer.
Five successive pulls for each animal in the study group were
tried by a person unaware of their treatment status. The mean
of all the values was taken and processed for statistical anal-
ysis. The values are expressed in the pound.
Spatial Memory—Continuous Alternation Test
Continuous alternation in a single session was measured using
a computerized Y-maze (TSE, Germany) to assess short-term
spatial memory [45, 64]. The equipment consisting of three
arms (40 cm long × 15 cm wide × 30 cm high) converge in an
equilateral triangular central area with 15 cm at its longest
axis. Rat in each case was placed at the end of one arm and
allowed to move freely through the maze for 5 min. The se-
quence of entry in to each arm was recorded automatically.
Spatial memory has been defined as the number and the se-
quence of entries of rat in to the arm during 5 min. All the arms
look alike and no reinforcer, motivation, or punishment was
used in this set of experiment. Further, the sequence of arm
movements was calculated and the percentage of alternation is
calculated as the number of alternations (entries into three
different arms consecutively) divided by the total possible
alternations (the number of arms entered minus 2).

Passive Avoidance Response
The passive avoidance response was assessed in rats to mon-
itor the learning and memory deficits following the procedure
as described by Tota et al. [75]. Briefly, the rat was placed in
the lighted compartment of a shuttle box (Techno, India). A
guillotine door isolates the compartment into light and dark
chamber. After an acclimatization period of 30 s, the guillotine
door was opened. Soon after the entry of the rat into the dark
compartment, the door was shut down and a low-intensity foot
shock (0.5 mA for 10 s) was given. The duration of a trial was
270 s. The first trial was acquisition. Retention tests were
performed at various times (24, 48, and 72 h were considered
as first, second, and third retention trials respectively) after the
acquisition trial. The transfer latency time refers to the transfer
of animal from one compartment to another which has been
recorded in seconds. The criterion for learning was taken as an
increase in the transfer latency time on retention trials as com-
pared to the acquisition (first) trial. The shock was not given in
the retention trials to avoid reacquisition.
Neurochemical Studies
mRNA expression DA-D2 and M2 receptor genes in brain
regions of rats: mRNA levels of M2 (frontal cortex, hippo-
campus), D2 (corpus striatum) receptor gene in different brain
regions of rats have been determined by RT-PCR (ABI, USA)
using the glyceraldehydes 3-phosphated dehydrogenase
(GAPDH) for normalization. The cDNA was amplified by
RT-PCR using ABI SYBER green qPCR Kit (Thermo
Scientific, USA) on the 7900HT Fast Real-Time PCR
System running at 40 cycles following the protocol: one cycle
of denaturation (95 °C, 10s), followed by 40 cycles of dena-
turation (95 °C, 10s), annealing (60 °C, 10 s), and extension
(72 °C, 45 s). A final extension (72 °C, 5 min) was performed.
The primer sequences used for M2 [11], D2 [12], and GAPDH
[12] are mentioned below:
DA-D2 FP 5′-AAGCGCCG RP 5′-GGCAATGATACACT
AGTTACTGTCAT-3′ CATTCTGGT-3′
CHRM2 FP 5′-GGCAAGCAAGA RP GCCAACAGGATAGCCA
GTAGAATAAA-3′ AGATT-3′
GAPDH FP 5′-CGGGAAGCTTGT RP 5′-CCAGCATCACCCC
GATCAATGG-3′ ATTTGA-3′
Immunoexpression of proteins by western blotting: As de-
scribed previously [13], frontal cortex hippocampus and cor-
pus striatum tissues were homogenized in RIPA buffer and
centrifuged at 12,000×g (15 min 4 °C) to remove the insoluble
material. The supernatant was taken out carefully and the pel-
let discarded. The prepared samples contained 30 μg protein/
lane and separated on 10% SDS-PAGE. The proteins were
electroblotted onto nitrocellulose membranes followed by
Chandravanshi et al.
blocking using blocking buffer (5% BSA). The membrane
was incubated overnight at 4 °C with antibodies DA-D2,
TH, ChAT (CST), and PKC β-1 (Abcam) followed HRP-
conjugated secondary antibodies (Sigma, USA) (1:4000) and
detected by ECL western blotting detection kit (Pierce, USA).
A digital gel image analysis system (ImageQuant LAS 500)
was used for semi-quantification of immunoreactivity and
normalized by β-actin.
Assay of acetylcholinesterase activity: Activity of acetyl-
cholinesterase (AChE)was measured following the procedure
as described by Ellman et al. [19] using acetylthiocholine iodide
as a substrate and 5,5′-dithiobis-2 nitrobenzoic acid (DTNB) as
the coloring agent. Briefly, the reaction mixture of a final volume
of 1.0 ml contained phosphate buffers (0.1 M, pH 7.4), a post-
mitochondrial fraction of frontal cortex or hippocampus (con-
taining 15–20 μg protein), acetylthiocholine iodide (ACTI),
and DTNB (5 mM). The degradation of acetylthiocholine iodide
was measured at 412 nm, and the results are expressed as micro-
moles ACTI hydrolyzed/milligram protein.
Estimation of biogenic amines and their metabolites in
brain regions of rats: To determine the levels of DA (dopa-
mine), DOPAC (3, 4-dihydroxyphenylacetic acid), and HVA
(homovanillic acid) in the corpus striatum of rats, reversed-
phase high-performance liquid chromatography coupled with
an electrochemical detector (HPLC–ECD) was carried out
following the method described by Kim et al. [34] with minor
modifications. The HPLC system (Waters, Melford, USA)
was integrated with a high-pressure isocratic pump (515
HPLC Pump), a sample injector valve, C-18 reverse phase
column (250 × 4 mm, particle size 5 μm), and an electrochem-
ical detector (464 Pulsed electrochemical detector). The brain
tissue was weighed and transferred into the tube and the pre-
chilled homogenized buffer solution (0.1 M perchloric acid
and 3,4-dihydroxy benzylamine, an internal standard at a final
concentration of 25 ng/ml) was added at a volume of 1 ml/
100 mg sample. Brain tissues were homogenized and kept on
ice for 15 min. The homogenate was centrifuged at maximal
speed (36,000×g) at 4 °C for 10 min and the supernatant was
collected and transferred to a new sample tube. The superna-
tant was filtered through nylon filters (0.22 μm; Millipore,
USA). Then 20-μl volumes of the sample were injected into
a column with a mobile phase (pH 4.2) containing sodium
dihydrogen phosphate (0.15 M), ethylenediaminetetraacetic
acid (0.25 mM), sodium octyl sulfate (1.75 mM), and 4%
methanol at a flow rate of 1.5 ml/min. The concentrations of
DA and its metabolites in corpus striatum of rats were deter-
mined by using an electrochemical detector coupled with an
integrator. Chromatograms were analyzed with the help of
Empower2 software (Waters, Melford, USA) and the results
were expressed in nanograms/gram tissue weight.
Estimation of arsenic in frontal cortex, hippocampus, and
corpus striatum of rats: Arsenic levels were estimated in the
frontal cortex, hippocampus, and corpus striatum of rats
Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic and Dopaminergic System in Brain of Developing...
by using the hydride system 60 atomic absorption spec-
trophotometer (HSAAS, ZEEnit 700) following the meth-
od of Ballentine and Burford [6]. Briefly, 0.1 g of tissue
was weighed and digested in 2-ml mixtures of concentrat-
ed nitric acid (1 ml) and perchloric acid (1 ml) in the
Kjeldahl flask. The sample was digested over a sand bath
until it becomes yellow in color. If the color of the digest
was brown, more nitric acid and the perchloric acid mix-
tures were added and the process of oxidation was repeat-
ed. The residue was made up to the known volume of HCl
(3%) in deionized water and aliquots were used to esti-
mate the arsenic levels using an atomic absorption spec-
trophotometer. A calibration curve was constructed by
adding known amounts of arsenic standard (Sigma) to
calculate arsenic levels in brain regions, and data were
presented in nanograms/gram tissue weight.
Protein Estimation
Protein content in the samples was measured following the
procedure as described by Lowry et al. [41] using bovine
serum albumin as a reference standard.
Statistical Analysis
Statistical analysis of the data has been carried out using
Graph Pad Prism software. The data have been analyzed using
one-way analysis of variance (ANOVA) followed by
Newman–Keuls test to compare all pairs of columns. Values
are expressed as mean ± SEM and values up to p < 0.05 have
been considered significant.
Results
Effect on Body and Brain Weight
Perinatal (GD6–PD21) exposure to arsenic (2.0 or 4.0 mg/
kg body weight) caused a significant decrease in the body
weight (F(2,57) = 7.78, 21%, p < 0.05; 32%, p < 0.05) of
rats that was observed on PD22 as compared to controls
(Fig. 1a). The body weight remained decreased
(F(2,57) = 4.36, 8%, p > 0.05; 16%, p < 0.05) in arsenic-
exposed rats on withdrawal of arsenic exposure on PD45
as compared to respective controls (Fig. 1a). However,
changes in the body weight was not significant in rats
exposed to arsenic at a low dose (2.0 mg/kg body weight).
No significant change in the brain weight was observed in
rats perinatally (GD6–PD21) exposed to arsenic at any of
the doses (2.0 or 4.0 mg/kg body weight) on PD22 and
PD45 as compared to respective controls (Fig. 1b).
Neurobehavioral Studies
Effect on Development of Physical Milestones and Reflexes
Rat pups were observed once daily to examine their general
appearance, the physical condition, including moving activi-
ties and appearance of hair, nose, eyes, and limbs. There was
no mortality in rat pups in any of the treatment groups.
Further, there was no delay in the development of eye-open-
ing, pinna detachment, incisor eruption, and fur growth in
arsenic-exposed rat pups as compared to controls (data not
shown). Reflex parameters were tested to assess the effects
of arsenic exposure on pups.
Surface righting: Following a neurotoxic dose of arsenic
(2 or 4 mg/kg p.o.) in rat pups, there was a delay in the
development of offspring righting reflex compared to con-
trol animals at PD7. Arsenic, however, did not affect the
righting ability of pups at a lower dose. The development
of righting ability was significantly impaired by arsenic at
higher dose at PD7. The present study shows that surface
righting remained increased on PD1, 3, 5, and 7 in the
arsenic-exposed group than controls (Fig. 2a).
Negative geotaxis effects: The results showed that arsenic
did not hamper the ability of pups to show negative geotaxis
reflex at PD5, 10, and 15 (Fig. 2b).
Cliff avoidance: The cliff avoidance response (F(2,27) =
7.38, 23%, p < 0.05; 54%, p < 0.01) was found to be delayed
in arsenic-exposed rat pups as compared to control rat pups at
both doses (Fig. 2c).
Effect on Spontaneous Motor Activity
Perinatal exposure to arsenic impaired the different parameters
of motor activity that was studied on PD22 and PD45, and
results are summarized in Table 1. A significant decrease in
the distance traveled (F(2,15) = 44.69, 37%, p < 0.001; 49%,
p < 0.001), time moving (F(2,15) = 41.98, 23%, p < 0.001;
42%, p < 0.001), and an increase in resting time (F(2,15) =
41.98, 36%, p < 0.001; 66%, p < 0.001) were observed in rats
exposed to arsenic (2.0 or 4.0 mg/kg body weight) on PD22 as
compared to controls (Table 1). At perinatal arsenic exposure,
we found increased stereotypic counts (F(2,15) = 15.17, 42%,
p < 0.01; 72%, p < 0.001) in arsenic-exposed groups with re-
spect to the control group. Distance traveled (F(2,15) = 12.03,
17%, p < 0.05; 25%, p < 0.01), time moving (F(2,15) = 7.18,
17%, p < 0.05; 24%, p < 0.05), resting time (F(2,15) = 7.18,
18%, p < 0.05; 25%, p < 0.05), and stereotypic counts
(F(2,15) = 2.55, 21%, p > 0.05; 44%, p > 0.05) exhibit a trend
of recovery, but the changes remained persistent on withdraw-
al of arsenic exposure in developing rats on PD45 as com-
pared to respective controls (Table 1). The locomotor activity
results show that arsenic-exposed rats are characterized by a
hypo-activity compared with control rats.
 Chandravanshi et al.

Fig. 1 Body weight (a) and brain A – Body Weight

weight (b) of arsenic-exposed rats CONT As I As II
on PD22 or after the withdrawal
of arsenic exposure on PD45.
60
Values are the mean ± SEM of
five animals in each group for
brain weight and 20 animals for 50
body weight. Different from *

Body weight (g)

control group, *p < 0.05 and 40
**p < 0.01 were determined by
one-way ANOVA with Newman– 30
Keuls test
**
20 **

10

0
PD22 PD45

B – Brain Weight

1.6

1.2
Brain weight (g)

0.8

0.4

0
PD22 PD45

Effect on Grip Strength
Effect of arsenic on the muscular strength of forelimb muscles
was evaluated by grip holding the capacity of rats. A decrease
in the forelimb grip strength (F(2,15) = 77.07, 30%, p < 0.01;
40%, p < 0.01) was observed on PD22 in rats perinatally
(GD6–PD21) exposed to arsenic (2.0 or 4.0 mg/kg body
weight) as compared to controls (Fig. 3a), while a trend of
recovery of the grip strength was observed in arsenic-
exposed rats on withdrawal of exposure. Decreased grip
strength was more prominent in rats exposed to arsenic at
higher dose (F(2,15) = 6.35, 17%, p < 0.05) on PD45 as com-
pared to respective controls (Fig. 3a).
Effect on Spatial Memory—Continuous Alternation Test
Concerning spatial memory like behavior, arsenic admin-
istration reduced the percentage of alternation in arsenic-
exposed groups as compared to control. A significant de-
crease in the spatial memory (F(2,15) = 24.24, 35%, p <
0.001; 54%, p < 0.001) tested by a Y-maze was observed
in rats exposed to arsenic perinatally from GD6 to PD21
at both doses (2.0 or 4.0 mg/kg body weight) on PD22 as
compared to rats in the control group (Fig. 3b). Although
a trend of recovery from the spatial memory was observed
(F(2,15) = 7.68, 14%, p < 0.05; 33%, p < 0.01) in arsenic-
exposed rats on withdrawal of arsenic exposure, a de-
crease in spatial memory was found to persist in arsenic-
exposed rats on PD45.
Effect on Learning and Memory—Passive Avoidance Test
Perinatally arsenic-exposed rats demonstrated impairment
in memory as evidenced by a reduction of transfer latency
time for the dark chamber. Perinatal arsenic exposure to
mothers at both doses was caused a significant impair-
ment in memory of pups on PD22. The decrease in the
retention of memory was more pronounced in rats treated
with arsenic at a higher dose. However, impairment in the
retention of memory remained persistent in rats exposed
to arsenic at a higher dose (4.0 mg/kg body weight) even
on withdrawal of arsenic exposure on PD45 in compari-
son to respective controls (Fig. 3c).
Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic and Dopaminergic System in Brain of Developing...

Fig. 2 Results of A - Surface Righting B - Negative Geotaxis

neurodevelopmental reflexes in
CONT As I As II
offspring. Surface righting reflex 12 CONT As I As II
60
(a) and negative geotaxis (b) were 10
** 50
monitored beginning on PD1 and

Time (s)
8 ***

Time (s)
PD5, respectively, cliff avoidance **
40
*
6 30
(c) was monitored beginning on
PND4. Values are the mean ± 4 20
SEM of ten animals in each 2 10
group. Different from control 0 0
group, *p < 0.05, **p < 0.01, and PD-1 PD-3 PD-5 PD-7 PD5 PD10 PD15
***p < 0.001 were determined by
one-way ANOVA with Newman–
Keuls test CONT As I As II
C - Cliff Avoidance
10 **
9
8 *

7
6

Time (s)
5
4
3
2
1
0
CONT As I As II
PD4

Neurochemical Studies gene were assessed in different brain regions. Exposure

Effects of Chronic Arsenic Exposure in DA-D2 and CHRM2
mRNA Expression
In order to evaluate the effects of arsenic exposure on
receptor genes, mRNA levels DA-D2 and of CHRM2
to arsenic during the perinatal period caused a signifi-
cant decrease in the mRNA expression of DA-D2 recep-
tor gene (F(2,6) = 45.62, 44%, p < 0.001; 68%, p <
0.001) on PD22 as compared to controls (Fig. 4a).
Further, the decrease in the mRNA expression of
DAR-D2 receptor gene (F(2,6) = 11.35, 29%, p < 0.05;

Table 1 Perinatal exposure

(GD6–PD21) to arsenic and effect Control Treatment groups
on different parameters of
spontaneous motor activity of rats As I As II
on PD22 and PD45 (2 mg/kg) (4 mg/kg)

Postnatal day 22
Total distance traveled (cm) 2370 ± 145.3 1487 ± 54.93*** 1207 ± 24.72***
Resting time (s) 116.8 ± 5.09 197.8 ± 8.57*** 194.8 ± 6.74***
Stereotypic count (s) 27.17 ± 1.72 38.83 ± 2.99** 46.83 ± 2.72***
Time moving (s) 183.2 ± 5.09 140.2 ± 6.13*** 105.2 ± 6.74***
Postnatal day 45
Total distance (cm) 2123 ± 93.83 1737 ± 88.86** 1512 ± 86.73***
Resting time (s) 44.7 ± 13.38 175.3 ± 5.50* 192.5 ± 5.99**
Stereotypic count (s) 31.5 ± 2.24 37.83 ± 2.34 41.33 ± 4.21
Time moving (s) 155.3 ± 13.38 124.7 ± 5.50* 107.5 ± 5.99**

Different parameters of spontaneous motor activity studied of arsenic-exposed rats on PD22 or after the with-
drawal of arsenic exposure on PD45. Values are the mean ± SEM of six animals in each group. Different from
control group, *p < 0.05, **p < 0.01, and ***p < 0.001 were determined by one-way ANOVA with Newman–
Keuls test
 Chandravanshi et al.

Fig. 3 Neurobehavioral CONT As I As II

dysfunctions observed at PD22
(after the exposure) and PD45 A - Grip Strength B - Y maze - Continuous Alternation
(withdrawal of exposure) of age 400
of rats. a The forelimb grip 60
350
strength measured by grip 50
300 *

% of Alternation
strength meter. Percentage of
continuous alternation was 250 40 **
***

Pound (w)
assessed by Y-maze system (b).
200
Passive avoidance response in the 30
** ***
retention tests were performed at 150 ** 20
various times (24, 48, and 72 h, 100
respectively) after the acquisition 10
50
trial in arsenic-exposed groups
(c). Values are the mean ± SEM of 0 0
CONT As I As II CONT As I As II CONT As I As II CONT As I As II
five to six animals in each group.
Different from control group, PD22 PD45 PD22 PD45
*p < 0.05, **p < 0.01, and
***p < 0.001 were determined by C - Passive Avoidance Response
one-way ANOVA with Newman–
Keuls test Acquisition Retention 1 Retention 2 Retention 3
300 *
300
*
250 * * * *
250
*
Transfer Latency Time

Transfer Latency Time

200 200 * *

150 150

100 100

50 50

0 0
CONT As I As II CONT As I As II
PD22 PD45

44%, p < 0.01) was found to persist in arsenic-exposed Effect on the Protein Expression of TH, DA-D2 Receptor
rats even on withdrawal of arsenic exposure on PD45. (Corpus Striatum), ChAT, AChE, and PKCβ-1 (Frontal Cortex
However, the decrease in the mRNA expression was not and Hippocampus)
restored in arsenic-exposed rats.
To further explore the mechanism by which arsenic impairs
the cholinergic and dopaminergic system, we evaluated the
Effect on Muscarinic–Cholinergic Receptors and CHRM2 protein expression of the DA-D2 receptor, TH, ChAT, and
Receptor Gene in Frontal Cortex and Hippocampus PKCβ-1 by western blotting.
DA-D2 receptor and TH protein expression levels in the
Arsenic (2.0 or 4.0 mg/kg body weight) exposure during corpus striatum were analyzed. As observed in Fig. 5, expres-
perinatal from GD6 to PD21 caused a significant de- sion of DA-D2 receptor (F(2,6) = 11.87, 1.33-fold, p < 0.05;
crease in the expression of CHRM2 receptor genes in 1.63-fold, p < 0.01) and TH (F(2,6) = 32.98, 1.43-fold, p <
the frontal cortex (F(2,6) = 10.62, 38%, p < 0.01; 55%, 0.01; 2.03-fold, p < 0.001) proteins in the corpus striatum in
p < 0.001) and hippocampus (F(2,6) = 22.97, 39%, p < both arsenic-exposed groups were significantly lower than in
0.001; 59%, p < 0.001) of rats on PD22 as compared the control group on PD22. Moreover, there was no significant
to controls (Fig. 4b). While a trend of recovery was change observed in the expression of DA-D2 receptor protein
found in the mRNA expression of CHRM2 receptor at the lower dose of arsenic after the withdrawal of exposure as
genes both in frontal cortex and hippocampus, the compared to controls (Fig. 5a). Down-regulation of the DA-
changes remained significantly decreased in rats exposed D2 receptor was significantly continued at a higher dose of
to arsenic at a higher dose PD45 in comparison to con- arsenic (F(2,6) = 27.23, 1.78-fold, p < 0.01) on PD45. The
trols (Fig. 4b). expression of TH (F(2,6) = 26.79, 1.30-fold, p < 0.01; 1.51-
Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic and Dopaminergic System in Brain of Developing...

Fig. 4 Relative mRNA levels of A

DA-D2 receptor gene in the 1.2 CONT As I As II
corpus striatum (a) and CHRM2
receptor gene in frontal cortex and 1
hippocampus (b) of arsenic-

DA-D2 mRNA Levels

Relative to Controls
exposed rats on PD22 or after the 0.8 *
withdrawal of arsenic exposure
on PD45. Values are the mean ± 0.6 *** **
SEM of three animals in each
group. Different from control 0.4 ***
group, *p < 0.05, **p < 0.01, and
***p < 0.001 were determined by 0.2
one-way ANOVA with Newman–
Keuls test 0
PD22 PD45
Corpus Striatum
B
1.2
CHRM2 mRNA Levels Relative to Controls
1

0.8 **
**
** *** **
0.6 ***
***
0.4

0.2

0 PD22 PD45 PD22 PD45

Frontal Cortex Hippocampus

fold, p < 0.001) in corpus striatum remained significantly de- Densitometric analysis revealed that perinatal exposure to ar-
creased on withdrawal of arsenic exposure on PD45 as com- senic was decreased the expression of ChAT, an enzyme in-
pared to controls (Fig. 5b). volved in the synthesis of acetylcholine, a neurotransmitter in
Compared with the control, expression of PKC β-1 was frontal cortex (F(2,6) = 14.51, 1.26-fold, p < 0.05; 1.50-fold,
reduced by F(2,6) = 32.84, 1.24-fold, 1.60-fold, in the hippo- p < 0.01) and hippocampus (F(2,6) = 16.01, 1.22-fold, p <
campus of rats in groups I and II, respectively, on PD22. 0.05; 1.56-fold, p < 0.01) on PD22 in comparison to controls
However, rats exposed to arsenic at a low dose exhibited a (Fig. 6b). In arsenic-exposed rats at the lower dose, expression
trend of recovery in the expression of PKC β-1 in hippocam- of ChAT was found to recover while the expression remained
pus on PD45 as compared to respective controls (Fig. 6a). significantly decreased in rats exposed to arsenic at a higher

Fig. 5 Relative protein A B

expression levels of DA-D2 51 kda TH 40 kda
DA-D2
receptor (a) and tyrosine
β-actin 42 kda β-acn 42 kda
hydroxylase (b) in the corpus
striatum of arsenic-exposed rats
on PD22 or after the withdrawal CONT As I As II
of arsenic exposure on PD45.
Each panel shows a representative 1.2
Western blot (top) and 1.2
1
densitometric analysis of bands in
the control and As-exposed *
0.8
Fold Change

groups. Values are the mean ± **

Fold Change

** 0.8 **
SEM of three animals in each ** ***
0.6
group. Different from control
***
group, *p < 0.05, **p < 0.01, and
0.4
***p < 0.001 were determined by 0.4
one-way ANOVA with Newman–
0.2
Keuls test
0 0
PD22 PD45 PD22 PD45
Corpus Striatum Corpus Striatum
 Chandravanshi et al.

Fig. 6 Relative protein

expression levels of PKC β-1 in A PKC β-1 77 kda
the hippocampus (a) and ChAT in β-acn 42 kda
the frontal cortex and
hippocampus (b) of arsenic- 1.2 CONT As I As II
exposed rats on PD22 or after the
1
withdrawal of arsenic exposure
on PD45. Each panel shows a ** *
0.8
representative western blot (top)

Fold Change
***
and densitometric analysis of 0.6
bands in the control and As-
exposed groups. Values are the 0.4

mean ± SEM of three animals in

0.2
each group. Different from
control group, *p < 0.05, **p < 0
0.01, and ***p < 0.001 were PD22 PD45
determined by one-way ANOVA Hippocampus
with Newman–Keuls test B
ChAT 68 kda
β-actin 42 kda
1.2

*
*
0.8 * **
**
Fold Change

**
0.6

0.4

0.2

0
PD22 PD45 PD22 PD45
Frontal Cortex Hippocampus

dose (4.0 mg/kg body weight) as compared to respective con- the acetylcholinesterase (AChE) activity was more
trols on PD45 (Fig. 6b). marked in rats exposed to arsenic at a higher dose
(4.0 mg/kg body weight).

Effect on Brain Acetylcholinesterase Activity in Frontal Cortex

and Hippocampus Effect on Dopamine and their Metabolites in Corpus Striatum

Acetylcholinesterase, an enzyme involved in the hydroly-
sis of acetylcholine, a neurotransmitter in the cholinergic
neurons that modulate the behavior, was studied in frontal
cortex and hippocampus of perinatally arsenic-exposed
rats. A significant decrease in the activity of acetylcholin-
esterase (AChE) was observed both in the frontal cortex
(F(2,12) = 10.03, 31%, p < 0.01; 48%, p < 0.001) and hip-
pocampus (F(2,12) = 8.97, 34%, p < 0.001; 45%, p <
0.001) of rats perinatally exposed to arsenic (2.0 or
4.0 mg/kg body weight) on PD22 as compared to controls
(Fig. 7). The decrease in the activity of acetylcholinester-
ase (AChE) was found to persist both in the frontal cortex
(F(2,12) = 4.20, 19%, p > 0.05; 25%, p < 0.01) and hippo-
campus (F(2,12) = 4.52, 13%, p > 0.05; 21%, p < 0.01)
even on withdrawal of arsenic exposure on PD45 as com-
pared to respective controls. The intensity of changes in
The levels of DA and their metabolites showed dose-
dependent change in the corpus striatum of arsenic-
exposed rats as compared to controls. A significant decrease
in the levels of striatal DA (F(2,9) = 32.79, 35%, p < 0.01;
60%, p < 0.001) and an increase in the levels of DOPAC
(F(2,9) = 43.78, 39%, p < 0.001; 53%, p < 0.001) and HVA
(F(2,9) = 18.76, 31%, p < 0.05; 64%, p < 0.001) were ob-
served in the corpus striatum of perinatally arsenic-exposed
rats as compared to controls on PD22. Both doses of arsenic
had persistent effects as a significant decrease in the levels of
DA (F(2,11) = 6.33, 28%, p < 0.05; 41%, p < 0.05) was ob-
served even after withdrawal of arsenic exposure. Higher
levels of DOPAC (F(2,11) = 8.84, 25%, p < 0.05; 36%, p <
0.01) and HVA (F(2,11) = 8.22, 16%, p > 0.05; 32%, p <
0.01) were found to persist on PD45 in comparison to respec-
tive controls (Fig. 8).
Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic and Dopaminergic System in Brain of Developing...

Fig. 7 Acetylcholinesterase CONT As I As II

10
activity in the frontal cortex and

μ mole Hydrolyzed / min / mg Protein

hippocampus of arsenic-exposed
rats on PD22 or after the with- 8
drawal of arsenic exposure on
*** **
PD45. Values are the mean ±
*** **
SEM of five animals in each 6
**
group. Different from control
group, **p < 0.01 and ***p < ***
0.001 were determined by one-
4
way ANOVA with Newman–
Keuls test 2

0 PD22 PD45 PD22 PD45

Frontal Cortex Hippocampus

Effects on Arsenic Levels in Brain Regions and varying period of exposure [5, 37, 89]. Still, there is
no study on the effect of perinatal arsenic exposure to cho-
The total arsenic levels in frontal cortex, corpus striatum, linergic and dopaminergic dysfunctions along with
and hippocampus of rats were measured on PD22 and neurodevelopmental consequences of developing brain of
PD45, and the results are presented in Table 2. A signif- rats. The previous studies have demonstrated that arsenic
icant increase in the levels of arsenic in the frontal cor- exposure might lead to cholinergic and dopaminergic dys-
tex, corpus striatum, and hippocampus was observed in functions of early life exposed rats at the same doses of the
perinatally arsenic-exposed rats on PD22 as compared to present study. The present study has succeeded in
controls (Table 2). However, the decrease in arsenic unveiling a mechanism of developmental neurotoxicity
levels was observed on withdrawal of arsenic exposure linking the arsenic levels in different brain regions of rats
in the frontal cortex, corpus striatum, and hippocampus and also assessing such changes are transient or persistent.
on PD45 as compared to rats exposed to arsenic imme- The reduction of body weight following perinatal exposure
diately on PD22. However, the increase in the arsenic to arsenic reported in the present study indicated that im-
levels remained persistent in selected brain regions at pairment of growth and general health status of rats is con-
both the doses of arsenic in comparison to respective sistent with earlier experimental reports [8, 15]. Decreased
controls on PD45 (Table 2). pattern of body weight remained to persist with a higher
dose of arsenic even after withdrawal of arsenic exposure,
suggesting that the effect is persisting and perinatal arsenic
Discussion exposure may have detrimental health effects.
It is found that arsenic exposure might cause to modu-
Many laboratories have reported that exposure to arsenic late the cognitive and motor functions even at low doses,
during the developing period of the brain in experimental and the changes might be associated with cholinergic and
animals induces brain changes and promotes neurotoxicity. dopaminergic adverse outcomes. With the result of the
These reports have explained the mechanism of arsenic- present study, we found the noticeable link between de-
induced neurotoxicity in animals at different ages, dose, velopmental arsenic exposure and motor or cognitive

Fig. 8 Relative dopamine and CONT As I As II

their metabolites (HVA, DOPAC) 7000
content in the corpus striatum of
arsenic-exposed rats on PD22 or 6000
ng/gm Tissue Weight

after the withdrawal of arsenic 5000

exposure on PD45. Values are the ** *
mean ± SEM of four to five 4000 ***
* **
animals in each group. Different 3000
from control group, *p < 0.05, *** *
***
**p < 0.01, and ***p < 0.001 2000
were determined by one-way 1000 * ***
ANOVA with Newman–Keuls **
test 0
PD22 PD45 PD22 PD45 PD22 PD45
DA HVA DOPAC
Corpus Striatum

Table 2 Perinatal exposure
(GD6–PD21) to arsenic and effect Brain regions/treatment groups
on arsenic levels in different brain
regions of rats Frontal cortex
Control
As I (2 mg/kg)
As II (4 mg/kg)
Hippocampus
Control
As I (2 mg/kg)
As II (4 mg/kg)
Corpus striatum
Control
As I (2 mg/kg)
As II (4 mg/kg)
Arsenic levels expressed as ng/g tissue weight. Values are the mean ± SEM of five animals in each group.
Different from control group, *p < 0.05, **p < 0.01, and ***p < 0.001 were determined by one-way ANOVA
with Newman–Keuls test
deficits in rats. Adverse health effects of arsenic on the
developing fetus and children are of particular attention to
the society and regulatory agencies. The effects of perina-
tal arsenic exposure on neurodevelopment consequence,
motor and cognitive function, including learning and
memory, in rats were evaluated. The timing of exposure
has no importance to the nature and the degree of the
observed neurotoxicity in an adult. In contrast, the devel-
oping brain will respond to a similar neurotoxic insult to a
different susceptibility stage of brain development. The
high sensitivity of developing rats compared with the
adults to the neurotoxicity of arsenic is most probably
due to the immaturity of detoxifying enzymes. Arsenic
may also interfere the maturation of BBB and receptors
of the brain during development, causing permanent dys-
functions [29, 57] that result in behavioral changes.
Growth delays and neural tube defects by the arsenic ex-
posure in human have been reported [2, 78], and the neuro-
logical and cognitive deficits in children were also observed
[36, 65, 81]. Negative geotaxis can assess the sensory input,
while cliff avoidance and surface righting assess motor func-
tion of rats. In the present study, developmental exposure to
arsenic impaired the negative geotaxis, cliff avoidance, and
surface righting ability of neonates. Corpus striatum, frontal
cortex, and hippocampus are believed to be involved in the
neurobehavioral reflexes [4, 83]. Further, neurochemical
changes in the brain can be predicted by impairment of these
reflexes and provide the basic mechanisms of brain damage
underlying the arsenic exposure.
It is well known that dopamine regulates the motor activity
and cognitive functions of rats and closely associated with
neurotransmission process. Levels of striatal dopamine are
necessary for the physiological function of the dopaminergic
system. TH is a rate-limiting enzyme for the synthesis of
Chandravanshi et al.
Postnatal day 22 Postnatal day 45
2.90 ± 0.54 2.07 ± 0.81
91.02 ± 9.41* 42.97 ± 17
334.20 ± 59.50*** 206 ± 29.38***
1.12 ± 0.24 0.82 ± 0.21
94.50 ± 22.23** 36.27 ± 8.23*
172.80 ± 24.92*** 109.60 ± 16.64***
4.82 ± 0.46 8.65 ± 0.72
416.50 ± 22.95*** 284.10 ± 40.77***
683.30 ± 12.80*** 442 ± 45.77***
dopamine [7]. Motor and cognitive functions and different
type receptor signaling are affected in rodents by the arsenic
exposure [35, 42, 72]. Therefore, we find out whether arsenic
exposure alters expression of TH protein in the corpus stria-
tum. Expression TH protein in the corpus striatum was signif-
icantly decreased in both arsenic exposure groups. In addition,
DA-D2 receptors in the striatum have a distinct role in the
regulation of motor function including locomotor activity.
To better understand the mechanism of underlying arsenic-
induced developmental neurotoxicity, we further observed
the declined protein expression of DA-D2 receptors as well
as mRNA level. Decrease striatal DA level was found in
arsenic-exposed rats in a dose-dependent manner, while in-
creased DOPAC and HVA levels were found in the corpus
striatum of rats treated with arsenic suggesting the dopaminer-
gic system as a target of arsenic exposure. DOPAC and HVA
are the main neuronal metabolites of dopamine; higher levels
of DOPAC and HVA contents are consistent with greater loss
of DA. These findings indicate that decreased dopamine level
may be related to the declined expression of TH in arsenic-
exposed rats at these low concentrations of arsenic during the
developmental period of the brain. These changes in the se-
lected endpoints of the dopaminergic system due to the accu-
mulation of arsenic in the corpus striatum might be related to
the behavioral or functional consequences. These findings
agree with the previously published reports, where transmis-
sion of dopaminergic neurons and its associated behaviors
were vulnerable to arsenic, which induced neurobehavioral
toxicity [88]. Hyperactivity and hypo-activity have also been
reported in arsenic-exposed rats, and these behaviors seemed
to be dependent on dose, route of exposure, and the period of
exposure to arsenic [12, 51].
Arsenic exposure has also been found to inhibit AChE
activity and ChAT expression in the brain of rats [17, 87],
Arsenic-Induced Neurotoxicity by Dysfunctioning Cholinergic and Dopaminergic System in Brain of Developing...
and it has also been reported that there is an inverse link
between arsenic exposure and plasma cholinesterase activity
in a human [3]. As most of these studies have been designed at
high doses of arsenic and in adult animals, the impact of low
doses of arsenic on the integrity of brain AChE activity during
the perinatal period of brain development is unexplored.
Further, declined frontocortical and hippocampal AChE activ-
ity, ChAT protein expression, as well as CHRM2 gene expres-
sion were observed in a dose-dependent manner. Inhibition in
the activity of acetylcholinesterase is a result of arsenic depo-
sition in the brain and is also associated with enhanced accu-
mulation of acetylcholine in the synaptic cleft and subsequent-
ly downregulation of cholinergic receptors both in the frontal
cortex and hippocampus. Accumulation of acetylcholine at
the synaptic cleft and arsenic in the frontal cortex and hippo-
campus of rats indicated vulnerability of the cholinergic sys-
tem to perinatal arsenic exposure. PKCβ-1 also plays a sig-
nificant role in the long-term potentiation and different pat-
terns of learning and memory [54, 86]. It can also be hypoth-
esized that decreased expression of PKCβ-1 in the hippocam-
pus was associated with impaired learning and memory in
arsenic-exposed rats in comparison to controls as observed
in the present study.
With these experiments, we also sought to define the cho-
linergic dysfunction patterns of the frontal cortex and hippo-
campus of developing rats as well as effects on learning and
memory induced by arsenic at low doses. Arsenic may affect
the integrity of muscarinic–cholinergic receptors and inhibit
intracellular signaling [56, 77]. They also defined the cogni-
tion and behavioral dysfunctions may occur due to the distur-
bances of the cholinergic system of the central nervous sys-
tem. The decline in learning and memory caused by the rela-
tively low doses of arsenic used here was accompanied by
distinct patterns of selected endpoints of the cholinergic sys-
tem. In our study, decrease in learning and memory is possible
due to the significant severity of cholinergic dysfunctions.
Furthermore, subjects with a decreased grip strength were
more likely to have a muscle weakness. This could be related
to the cholinergic dysfunctions in the frontal cortex and hip-
pocampus of rats. Impairment in learning and memory clearly
suggested that even low doses of arsenic will injure the frontal
cortex and hippocampus of developing rats. Cognitive impair-
ments in humans assessed after arsenic exposures have been
associated with Alzheimer’s disease and established the link
between arsenic and Alzheimer’s disease [24]. However, peri-
natal arsenic exposure is thought to inhibit the cholinergic
system in the frontal cortex and hippocampus, along with
cognitive deficits of developing rats at these low doses. It is
well established that arsenic crosses the placenta and blood–
brain barrier; therefore, exposure may occur from the begin-
ning of life and accumulate in the different brain regions. An
increase of arsenic levels found in the corpus striatum, frontal
cortex, and hippocampus of perinatal arsenic-exposed rats
could be associated with the cholinergic and dopaminer-
gic dysfunctions. We investigated a mechanism involved
in arsenic-induced developmental neurotoxicity. These in-
clude modification in dopaminergic and cholinergic sig-
naling, and behavioral deficits, including locomotion
learning and memory. This rodent model that may even-
tually be useful for understanding arsenic-induced neuro-
logical deficits in children during the duration of perinatal
exposure. Dysfunction of both dopaminergic and cholin-
ergic systems has been alarmed in a different type of
neurological disorder characterized by motor and cogni-
tive deficits including learning and memory. In the present
study, functional deficits in spatial learning, memory, and
motor activity following arsenic exposure at high and low
doses made it possible to significantly cause dopaminer-
gic and cholinergic dysfunctions in perinatally exposed
rats. A trend of recovery was observed in selected behav-
ioral and neurochemical endpoints after 23 days of with-
drawal of arsenic exposure; however, most of these
changes remained persistent. It is further suggested that
the intensity of these changes could be more harmful in
case exposure to arsenic occurs during the developmental
period of the brain for the reason that development of
dopaminergic and cholinergic receptors in the brain con-
tinues from perinatal to early life periods of rats.
In conclusion, our findings indicate that perinatal arsenic
exposure leads to delay and disturbance of neurodevelopment.
Taking into account the possibility that arsenic might inhibit
the normal development of dopaminergic and cholinergic neu-
rons in the developing brain when exposed during the perina-
tal period, developmental neurotoxicity cannot be excluded.
Further, developmental exposure to arsenic may be a risk fac-
tor for increased vulnerability to neurological disorders, and
studies are needed in order to progress the urgently needed
risk assessment. In this light, our findings are relevant: they
suggest that dopaminergic and cholinergic dysfunctions with
neurobehavioral deficits represent key issues that may be as-
sociated with accumulation of arsenic in different brain re-
gions. These findings put forth a new perspective on how
environmental arsenic exposure affects the pathogenesis of
neurodevelopmental disorders in children. The underlying
mechanisms of these shared changes may provide an avenue
for novel treatments and address preventive strategies for
arsenic-induced neurodevelopment disorders in children.
Acknowledgements: The authors thank the Director of the CSIR–Indian
Institute of Toxicology Research (CSIR-IITR), Lucknow for his support
and keen interest in the present study. Financial support by the University
Grants Commission, New Delhi for carrying out the study is gratefully
acknowledged.
Compliance with Ethical Standards
Conflict of Interest The authors state no conflict of interest.

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