Neurochemistry International 93 (2016) 51e63

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Neurochemistry International
journal homepage: www.elsevier.com/locate/nci

Brain cholinergic alterations in rats subjected to repeated

immobilization or forced swim stress on lambda-cyhalothrin exposure
Rajendra K. Shukla a, b, Richa Gupta a, Pranay Srivastava a, Yogesh K. Dhuriya a,
Anshuman Singh a, Lalit P. Chandravanshi a, Ajay Kumar b, M. Haris Siddiqui c,
Devendra Parmar a, Aditya B. Pant a, Vinay K. Khanna a, *
a
Developmenatl Toxicology Laboratory, Systems Toxicology & Health Risk Assessment Group, CSIR e Indian Institute of Toxicology Research, Post Box 80,
Vishvigyan Bhawan, 31 MG Marg, Lucknow 226 001, India
b
Department of Biochemistry, Integral Institute of Medical Sciences & Research, Integral University, Lucknow 226 026, India
c
Department of Bioengineering, Faculty of Engineering, Integral University, Lucknow 226 026, India

a r t i c l e i n f o a b s t r a c t

Article history: Role of immobilization stress (IMS), a psychological stressor and forced swim stress (FSS), a physical
Received 23 July 2015 stressor was investigated on the neurobehavioral toxicity of lambda-cyhalothrin (LCT), a new generation
Received in revised form type-II synthetic pyrethroid. Pre-exposure of rats to IMS (15 min/day) or FSS (3 min/day) for 28 days on
3 December 2015
LCT (3.0 mg/kg body weight, p.o.) treatment for 3 days resulted to decrease spatial learning and memory
Accepted 28 December 2015
Available online 31 December 2015
and muscle strength associated with cholinergic-muscarinic receptors in frontal cortex and hippocampus
as compared to those exposed to IMS or FSS or LCT alone. Decrease in acetylcholinesterase activity,
protein expression of ChAT and PKC-b1 associated with decreased mRNA expression of CHRM2, AChE and
Keywords:
Immobilization stress
ChAT in frontal cortex and hippocampus was also evident in rats pre-exposed to IMS or FSS on LCT
Forced swim stress treatment, compared to rats exposed to IMS or FSS or LCT alone. Interestingly, changes both in behavioral
Lambda-cyhalothrin and neurochemical endpoints were marginal in rats subjected to IMS or FSS for 28 days or those exposed
Brain cholinergic deficits to LCT for 3 days alone, compared to controls. The results suggest that stress is an important contributor
Impaired learning in LCT induced cholinergic deficits.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction play an important and crucial role in modulating the cognitive

Stress is a common experience of life to which a significant
population is exposed world over in one form or the other. Increase
in the release of corticosteroids due to hypothalamic-pituitary-
adrenal axis (HPA e axis) activation as a result of stress is one of
the potential mechanisms that could affect the physiological ho-
meostasis and pharmacological functions (Herman and Seroogy,
2006; Xiong and Zhang, 2013). Stress induced changes in the
functioning of neurotransmitters including cholinergic system have
been reported both in the affected individuals and experimental
animals (Conrad, 2010; Wang et al., 2009). Alterations in acetyl-
cholinesterase activity and cognitive deficits as a result of stress
have been observed (Das et al., 2005; Rao and Raju, 2000; Lee et al.,
2009). Repeated stress can affect the hippocampal neurons which
* Corresponding author.
E-mail address: vkkhanna1@gmail.com (V.K. Khanna).
functions (Fuchs and Flügge, 1998; Sapolsky, 2000). Disruption in
the blood brain barrier (BBB) permeability due to stress is another
mechanism demonstrated by a number of investigators in experi-
mental studies (Abdel-Rahman et al., 2002; Friedman et al., 1996;

Skult tyova
e  et al., 1998). The severity of changes in the BBB
permeability however, depends on the type, degree and duration of
stress (Hanin, 1996; Fuchs and Flügge, 1998; Abdel-Rahman et al.,
2002). A number of chemicals which do not cross the BBB in normal
situations could easily reach the brain under stress (Abdel-Rahman
et al., 2002; Friedman et al., 1996) Reports of psychological dis-
turbances, mood and brain related disorders in veterans returned
after the Persian Gulf War led to the realization that stress can
significantly contribute to enhance the toxicity of chemicals used
either as warfare agents or prophylactic agents (Baker et al., 1997;
Binns et al., 2008; Golomb, 2008; Parihar et al., 2013).
Amongst different classes of pesticides, synthetic pyrethroids
are preferred world over for their use in controlling large number of
insects and pests due to high bioefficacy both in occupational and

http://dx.doi.org/10.1016/j.neuint.2015.12.012
0197-0186/© 2016 Elsevier Ltd. All rights reserved.
52 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63
non-occupational settings (Soderlund et al., 2002; Wolansky and
Harrill, 2008). As a result, a number of formulations of pyre-
throids have been developed in recent years which have low
toxicity and can be put into practice (Bouwman et al., 2006; Turgut
et al., 2011). LCT, a new generation type II synthetic pyrethroid has
extensive uses both alone and also in combination with other
pesticides to control a large number of insects and pests in agri-
culture, home gardens and green houses (He et al., 2008; Righi and
Palermo-Neto, 2003). Because of its potential to control disease
vectors and ectoparasites, LCT is used frequently in public health
programmes and veterinary practices (Fetoui et al., 2008). Presence
of LCT residues in vegetables, cattle meat and milk enhances the
risk of human exposure to it (McCauley et al., 2006; Scollon et al.,
2011; Wu et al., 2013; Yadav et al., 2015). In population based
biomonitoring studies in USA, Germany and China, high levels of
metabolites detected in the urine further reveals the risk of human
s-Alcala
exposure to pyrethroids (Barr et al., 2010; Quiro  et al., 2014;
Wu et al., 2013). Recently, in a mother-child cohort study, low level
exposure to deltamethrin, a type II synthetic pyrethroid was found
to cause cognitive deficits in children (Viel et al., 2015). The
cognitive deficits in exposed children were associated with high
levels of cis-DBCA, a selective metabolite of deltamethrin and 3-
PBA, a general metabolite of pyrethroid in the urine of these chil-
dren (Viel et al., 2015). Cognitive deficits following low level py-
rethroid exposure have also been observed in experimental studies
with greater vulnerability in young animals (Ansari et al., 2012a;
Nasuti et al., 2013; Hossain et al., 2004). Of different mechanisms
suggested, increased ER stress and apoptosis associated with
impaired neurogenesis and choniergic dysfunctions in hippocam-
pus and alterations in brain neurotransmitter levels were associ-
ated with learning deficits (Nasuti et al., 2013; Hossain et al., 2015).
Impairment in learning and memory on early life exposure to LCT
was also observed in developing rats in an earlier study by us
(Ansari et al., 2012a). Interestingly, learning and memory deficits
were associated with brain cholinergic dysfunctions due to
enhanced oxidative stress (Ansari et al., 2012a).
Since humans encounter stress in routine life (though stressors
may be different), exposure to environmental chemicals can occur
even at low doses. The magnitude of exposure to chemicals how-
ever, may differ and largely depends on settings and situations. In
view of the different situations that may be encountered in real life,
different combinations of stressors have been used to assess their
impact on the neurotoxic potential of chemicals and drugs (Song
et al., 2002; Habr et al., 2014; Jortner, 2008; Roig et al., 2006).
However, subchronic effect (4 weeks/28 days) of stress on the
neurotoxicity of chemicals has been carried out extensively (Abdel-
Rahman et al., 2002, 2004; Parihar et al., 2013; Terçariol et al.,
2011). Recently, it was observed by us that pre-exposure to IMS, a
psychological stressor or FSS, a physical stressor for 28 days
resulted in increase of plasma corticosterone levels in rats treated
with LCT for 3 days (Shukla et al., 2015). Enhanced BBB permeability
associated with alterations in the levels of neurotransmitters in the
brain regions was also observed in rats pre-exposed to either of the
stressors on LCT exposure. Interestingly, no significant change in
any of the parameters was observed in rats exposed to IMS, FSS or
LCT alone.
In view of enhanced risk of LCT exposure and associated health
effects, the present study has been carried out to assess the sub-
chronic effect of IMS or FSS on LCT treatment on the integrity of
cholinergic-muscarinic receptors and other key parameters asso-
ciated in modulating the integrity of brain cholinergic system in
rats. As role of brain cholinergic receptors is well accepted to
modulate the learning and memory, both Y-maze and passive
avoidance test have been employed to assess the effect on the
spatial memory and learning respectively (Jamal et al., 2007; Ansari
et al., 2012a; Sankhwar et al., 2012; Yadav et al., 2011). The dose of
LCT (3 mg/kg body weight for 3 days) used was non-toxic and the
selection of dose was based on the pilot studies carried out by us as
repeated exposure of rats to LCT (1 or 3 mg/kg body weight for 3
days) had no significant effect on the binding of brain cholinergic-
muscarinic receptors and other cholinergic functions.
2. Materials and methods
2.1. Animals and experimental procedure
Adult male rats of Wistar strain (200 ± 20 gm) procured from
the animal breeding colony of CSIR e Indian Institute of Toxicology
Research (CSIR-IITR), Lucknow were used in the present study. Rats
were housed in polypropylene cages in the experimental room in
standard hygienic conditions. The experimental animal rooms had
12 h light/dark cycle and the temperature was set at 25 ± 2
C. Rats
had free access to food and water and were acclimatized for seven
days before the start of the experiment. The animals were
randomly divided in to six treatment groups containing 31 rats
each and received one of the assigned treatments as per mentioned
details. In the first group, rats were handled daily and given corn oil
orally once daily for three days (Day 26, 27 and 28) and served as
controls. Rats in the second group were handled daily and exposed
to LCT (3.0 mg/kg body weight, p.o. suspended in corn oil) once
daily for three days (Day 26, 27 and 28). In the third group, rats
were subjected to IMS once daily for 28 days by placing them in to
acrylic tubes (22 cm length and 7 cm diameter; 1 session of 15 min/
day) following the procedure as described by Rao et al. (2001). Rats
in the fourth group were subjected to IMS once daily for 28 days
identically as in group III and exposed to LCT (3.0 mg/kg body
weight, p.o. suspended in corn oil) for three days (Day 26, 27 and
28) as in group II. In the fifth group, rats were subjected to FSS once
daily for 28 days by placing them individually in a glass cylinder
(45 cm high, 20 cm in diameter) filled with water (temperature
around 25 ± 2
C) up to a height of 30 cm (1 session of 3 min/day)
following the procedure as described by Badowska-Szalewska et al.
(2009). Rats in the sixth group were subjected to FSS once daily for
28 days identically as in group V and exposed to LCT (3.0 mg/kg
body weight, p.o. suspended in corn oil) for three days (Day 26, 27
and 28) identically as in group II.
The treatment of LCT and stress procedure was carried out be-
tween 09:00e12:00 h. LCT was suspended in corn oil and admin-
istered orally (once daily for three days e Day 26, 27 and 28) in the
assigned groups. Incase of co-exposure to stressors and LCT, rats
were first subjected to either IMS or FSS. These rats were treated
with LCT (p.o) 15 min after stress exposure. There was no elimi-
nation of rats from any of the treatment groups. 24 h after the last
exposure, separate set of 5 rats from each treatment group was
used for assessment of behavior. Separate set of 5 rats were used for
the assay of cholinergic-muscarinic receptors and AChE activity
following the procedure as described in detail. For qRT-PCR and
Western blotting, separate set of 3 rats from each treatment group
was used. Rats were sacrificed by cervical decapitation and their
brains removed immediately and dissected into frontal cortex and
hippocampus following the standard procedure (GItiwinski and
Iversen, 1966). The experimental protocol was approved by the
Institutional Animal Ethics Committee (IAEC) of CSIR-IITR, Lucknow
and all experimental procedures were carried out in accordance
with the guidelines laid down by the Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA),
Ministry of Environment and Forests (Government of India), New
Delhi, India.
 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63
2.2. Behavioral studies
2.2.1. Grip strength
A computerized grip strength meter (TSE, Germany) was used to
assess the fore limb grip strength in control and treated rats
following the standard procedure as described by Yadav et al.
(2009). Briefly, rats were gently held from the neck and base of
the tail. The forelimbs of the rats were kept on the bar of the grip
strength meter and rats were pulled back gently. The reading was
automatically recorded on the computer soon the rat released the
bar. Five successive pulls were tried for each rat by a person not
aware of their treatment status. The mean of all the values was
taken and processed for statistical analysis.
2.2.2. Learning and memory ability
Effect on learning and memory in rats treated with LCT or those
subjected to stress alone or on their co-exposure with LCT and
stress was assessed by passive avoidance and Y maze test.
2.2.2.1. Passive avoidance test. The passive avoidance response in
rats was assessed using a shuttle box consisting of light and dark
compartment (Techno, India) following the standard procedure as
described by Yadav et al. (2011). Briefly, the transfer of rat from the
light to dark compartment was recorded as transfer latency time
(TLT) in seconds. The first trial was for the acquisition and retention
was tested in subsequent trials, 24 h after the first trial. The crite-
rion for improved cognitive activity was taken as increase in the TLT
on retention trials (second trial and further) as compared to
acquisition trial (first trial).
2.2.2.2. Y-maze. Spatial memory was assessed using Y-maze (TSE,
Germany) following the procedure as described by Wang et al.
(2009). Out of three arms of the Y-maze, one arm was designated
as start arm followed by novel arm and other arm based on the
extra-maze cues easily distinguished by the animals. One large
painted wooden object which could be distinguished by texture
and paint scheme was placed at the end of each arm as an intra-
maze cue. As the object was heavy, rats could not move it. Briefly,
during the training session, rats were kept in the start arm and
permitted to explore the start arm and other arm for a period of
15 min. The novel arm was blocked during this time. After a period
of 4 h, the novel arm was opened and rats were again permitted to
explore all the three arms for 5 min. It was ensured that the spatial
location of the objects at the end of the arms was same with respect
to the extra-maze cues. The start arm was not included in analysis
because the rats were placed there at the beginning of the testing
trial to avoid any bais. As the front paws of the rats crossed into an
arm, the entry to the arm was scored positive. The results are
expressed as % of time spent and % of entries in novel arm versus
other arm.
2.3. Neurochemical studies
2.3.1. Neurotransmitter receptor binding assay
Assay of cholinergic muscarinic receptors in frontal cortex and
hippocampus was carried out by radioligand receptor binding
following the standard procedure (Khanna et al., 1994). Crude
synaptic membrane was prepared by homogenizing the frontal
cortex and hippocampus individually in 19 volumes of Tris-HCl
buffer (5 mM, pH 7.4). The homogenate was centrifuged at
40,000  g for 15 min at 4
C and the sedimented pellet washed
twice by resuspending in Tris-HCl buffer (5 mM, pH 7.4) followed by
recentrifugation (40,000  g for 15 min at 4
C). The pellet was
finally suspended in Tris-HCl buffer (40 mM, pH 7.4) and stored
at 20
C. Briefly, the final volume of the reaction mixture was 1 ml
53
that contained Tris-HCl buffer (40 mM, pH 7.4), membrane protein
(300e400 mg) and 3H-QNB (42 Ci/mmole, Perkin Elmer, USA,
1 X 109 M) as a radioligand. The binding tubes in triplicate were
incubated for 15 min at 37
C in the presence or absence of atropine
sulphate (1 X 106 M). Soon after the incubation, the contents from
the binding tubes were rapidly filtered on glass fiber discs (25 mm
diameter, 0.3 mm pore size, Whatman GF/B) over vacum. The filter
discs were washed twice with cold Tris-HCl buffer (40 mM, pH 7.4)
to remove the unbound radioligand. The filter discs were dried and
transferred in to glass scintillation vials followed by addition of
scintillation mixture (PPO, POPOP, naphthalene, toluene and
methanol). The vials were counted using b-scintillation counter
(Packard, USA) at an efficiency of 30e40% to determine the mem-
brane bound radioactivity. Specific binding was calculated by sub-
tracting the non-specific binding (in the presence of atropine
sulphate) from the total binding (in the absence of atropine sul-
phate) and has been expressed as pmoles 3H-QNB bound/gm pro-
tein. Scatchard analysis was carried out using different
concentrations of 3H-QNB (1/10e10 times of the affinity of radio-
ligand) to determine whether change in the binding is due to
alteration in the affinity (Kd) or number of receptor binding sites
(Bmax).
2.3.2. Assay of brain acetylcholinesterase activity
Assay of acetylcholinesterase (AChE) activity in frontal cortex
and hippocampus was carried out following the standard method
(Ellman et al., 1961) using acetylthiocholine iodide as a substrate
and 5, 50 -dithiobis-2 nitrobenzoic acid (DTNB) as the coloring
agent. The reaction mixture in a final volume of 1.0 ml contained
phosphate buffer (0.1 M, pH 7.4), post-mitrochondrial fraction
(15e20 mg protein) from frontal cortex or hippocampus, acetylth-
iocholine iodide (154.8 mM) and DTNB (5 mM). The degradation of
acetylthiocholine iodide was measured at 412 nm and results are
expressed as mmol acetylthiocholine iodide hydrolysed/min/mg
protein.
2.3.3. Expression of neurotransmitter receptor genes
The mRNA expression of muscarinic-cholinergic receptors
(CHRM2), ChAT and AChE in frontal cortex and hippocampus was
carried out following the standard procedure (Shah et al., 2008;
Singh et al., 2014). Briefly, RNA extraction and cDNA synthesis
was carried out in isolated brain regions (frontal cortex/hippo-
campus) in 1 ml of ice-cold TRIzol (Invitrogen, Carlsbad, CA, USA)
and homogenized with hand homogenizer. For chloroform
extraction and isopropyl alcohol precipitation, RNA was dissolved
in RNAse free water (50 ml). To check the purity, RNA (0.5 mg) was
loaded onto a 1% agarose gel, subjected to electrophoresis, stained
with ethidium bromide and visualized by UV transilluminator. The
RNA concentration and purity was measured on a NanoDrop ND-
1000 (Thermo Scientific, Wilmington, DE, USA). Subsequently, the
cDNA was synthesized from total RNA using the high capacity cDNA
reverse transcription kit (Applied Biosystems, USA) according to the
recommended protocol. The tube was placed into the thermal
cycler that was set at standard conditions (step I e 25
C for 10 min,
step II e 37
C for 120 min, step III e 85
C for 5 min and step IV e
∞). For quantitative PCR, cDNA synthesized by High-Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, USA) was used and
analyzed by qRT-PCR (ABI 7900HT, Applied Biosystems, USA) using
SYBR green dye (Applied Biosystems, USA) following the standard
protocol. The sequence of primers used mentioned below has been
described earlier (Borges et al., 2001; Madziar et al., 2008; Morley
et al., 2004).
(1) CHRM2 FP 50 -GGCAAGCAAGAGTAGAATAAA-30 and
54 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63
RP 50 - GCCAACAGGATAGCCAAGTG-30 ;
(2) ChAT FP50 -CGGGATCCTGCCTCATCTTCTCTGGTGT-30 and
RP 50 - GGCGGAATTCAATCACAACATC-30 ;
(3) AChE FP 50 -GCTCACGTAGATTTATGCCACCAGA-30 and
RP 50 -TTGATCCAGCAGGCCTACATTG 30 ;
(4) b-actin FP 50 -CGTGGGCCGCCCTAGGCACCA-30 and
RP 50 -GGGGGGACTT GGGATTCCGGTT-30 .
The PCR conditions were the same as per approved protocol
(50
C for 2 min, 90
C for 10 min, 95
C for 0.15 s and 60
C for
1 min). b-actin was used as an endogenous control to normalize the
data. Alteration in mRNA has been referred as Relative Quantifica-
tion as compared to controls.
2.3.4. Expression of choline acetyltransferase and PKC b-1 proteins
Expression of choline acetyltransferase (ChAT), a marker for
cholinergic neurons and PKC b-1 in frontal cortex and hippocampus
was assessed by Western blotting (Jamal et al., 2007). Briefly, the
frontal cortex/hippocampus was homogenized individually in RIPA
buffer containing TriseHCl (50 mM, pH 6.8), NaCl (150 mM), so-
dium deoxycholate (0.5%), SDS (0.1%), protease inhibitor and
Triton-100X (1%) and centrifuged at 12,000  g (15 min, 4
C) to
remove the insoluble material. The pellet was discarded and the
supernatant further mixed with laemmli buffer containing
TriseHCl (60 mM, pH 6.8), SDS (2%), glycerol (10%), b-mercaptoe-
thanol (5%), bromophenol blue (0.01%) and boiled for 5e7 min. The
sample (30 mg protein/lane) was electrophoresed on SDSePAGE
(12%), electroblotted on to nitrocellulose membrane (Millipore,
USA) and blocked with blocking buffer (Western blocker solution
TM Sigma, USA). The membrane was washed and incubated with
primary antibody (Anti-PKC b-1, Sigma, USA, 1:1000 and Anti-ChAT,
Sigma, USA, 1:1000 dilution) for 24 h at 4
C followed by incubation
with HRP linked secondary antibody (anti-mouse IgG, 1:2000) at
room temperature for 1 h. The blot was washed and developed
using an immobilon western chemiluminescent HRP substrate
(Millipore, USA) following the recommended procedure once the
incubation was over. b-Actin was probed as an internal control and
used to confirm that an equal amount of protein was loaded in each
lane. A digital gel image analysis system (Image Quant LAS 500, GE
Healthcare Life Sciences, US) was used for semi-quantification of
protein.
2.3.5. Protein estimation
Protein content in the samples has been determined following
the method of Lowry et al. (1951) using bovine serum albumin as a
reference standard.
2.4. Statistical analysis
In the present study, two-way ANOVA was carried out using
GraphPad prism (3.0), USA software to analyze the changes in
behavioral and neurochemical endpoints on exposure to the
stressor (IMS or FSS) or LCT and to assess the interaction in each
brain regions. Further, the general linear regression model has been
used to assess the effect level of IMS, FSS and LCT. When interaction
was found significant, one way ANOVA was used to compare among
the groups. Bonferroni post hoc test was carried out to assess the
level of significance. The data has been expressed as mean ± SEM
and values up to p < 0.05 are considered statistically significant.
3. Results
3.1. Behavioral studies
3.1.1. Effect on grip strength
The two-way ANOVA revealed that there was no significant
interaction (F2,12 ¼ 0.60, p > 0.05) between LCT and stress exposure
while LCT (F1,12 ¼ 24.14, p < 0.001) and stress (F2,12 ¼ 5.45, p < 0.05)
could affect the fore limb grip strength (Fig. 1). The regression
model revealed that LCT 185 {95%C.I: 93e277, p < 0.001}, IMS 205
{95%C.I: 92e317, p < 0.001} and FSS 143 {95%C.I: 30e256, p < 0.01}
caused significant decrease in the grip strength as compared to
control.
3.1.2. Effect on learning and memory
3.1.2.1. Effect on passive avoidance response e assessed by suttle box.
Although, exposure to LCT (F1,12 ¼ 16.43, p < 0.01) and stress
(F2,12 ¼ 5.56, p < 0.05) modified the TLT on the third retention trial
as assessed by two-way ANOVA, no interaction (F2,12 ¼ 2.26,
p > 0.05) was observed between LCT and stress exposure. The
regression model showed that LCT 90 {95%C.I: 35e146, p < 0.01},
IMS 62.5 {95%C.I: 5.50e130, p < 0.05} and FSS 70 {95%C.I: 2.8e138,
p < 0.05} had significant decrease in the TLT on the third retention
trial as compared to control (Fig. 2A).
3.1.2.2. Effect on spatial learning and memory e assessed by Y maze.
Although, exposure to LCT (F1,12 ¼ 12.11, p < 0.01) and stress
(F2,12 ¼ 5.19, p < 0.05) modified the TLT on the percentage of entries
in novel arm as assessed by two-way ANOVA, no interaction
(F2,12 ¼ 0.98, p > 0.05) was observed between LCT and stress
exposure. The regression analysis further showed that LCT 6.36
{95%C.I: 2.08e10.6, p < 0.01}, IMS 8.5 {95%C.I: 3.26e13.7, p < 0.01}
and FSS 7.04 {95%C.I: 1.80e12.2, p < 0.05} caused significant
decrease in the percentage of entries in the novel arm as compared
to controls (Fig. 2B).
There was no significant interaction (F2,12 ¼ 0.80, p > 0.05) be-
tween LCT and stress exposure but stress (F2,12 ¼ 3.41, p < 0.05) and
LCT (F1,12 ¼ 8.27, p < 0.05) had significant effect on the percentage
of time spent in novel arm while the data was analyzed by two-way
ANOVA (Fig. 2C). The regression analysis showed that LCT 3.80 {95%
C.I: 0.51e7.09, p < 0.05}, IMS 3.9 {95%C.I: 0.08e7.96, p > 0.05} and
FSS 2.55 {95%C.I: 1.47e6.57, p > 0.05} caused significant decrease in
1400 CONT LCT
1200
1000
800 *a *b
Pound
600
400
200
0
No Stress IMS FSS
Fig. 1. Effect on grip strength following repeated exposure of rats to stressors for 28
days and lambda-cyhalothrin for 3 days. Values are mean ± SEM of five rats in each
group. The data was analyzed by two-way ANOVA followed by Bonferroni test. LCT
exposure decreased grip strength in rats subjected to IMS (p < 0.05) or FSS (p < 0.05),
compared to those exposed to IMS or FSS alone respectively. There was no significant
interaction (p > 0.05) between LCT and stress on the grip strength. a-compared to IMS
group, b-compared to FSS group. CONT e Control, LCT e Lambdaecyhalothrin, IMS e
Immobilization stress, FSS e Forced swim stress.
 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63 55

A. Shuttle box - Passive avoidance response

300 Acquisition Retention 1 Retention 2 Retention 3

Transfer latency time (sec)

250 ***a ***a ***a ***a ***a ***a
***a ***a ***a ***a ***a
***a
200

150

100

50

0
CONT LCT IMS IMS+LCT FSS FSS+LCT

B. Y Maze - Percentage of entries C. Y Maze - Percentage of time spent

50 Novel Arm Other Arm
50 Novel Arm Other Arm

40
40
*b **b **b *b *b
**b *b
% Enteries

% Time spent
30
30 ***b

20 20

10 10

0 0
CONT LCT IMS IMS+LCT FSS FSS+LCT CONT LCT IMS IMS+LCT FSS FSS+LCT

Fig. 2. Effect on learning and memory assessed by shuttle box and Y-maze following repeated exposure of rats to stressors for 28 days and lambda-cyhalothrin for 3 days. Values are
mean ± SEM of five rats in each group. The data was analyzed by two-way ANOVA followed by Bonferroni test. There was no significant effect in retention trials on LCT exposure in
rats subjected to IMS or FSS as compared to acquisition trial (A). LCT exposure resulted to affect percentage of entries (B) and time spent in the novel arm vs other arm (C) in rats
subjected to IMS or FSS assessed by Y maze. No significant interaction (p > 0.05) between LCT and stress was observed in any of the tests. a-compared to acquisition trial, b-
compared to novel arm. Significantly differs (*p < 0.05, **p < 0.01, ***p < 0.001). CONT e Control, LCT e Lambdaecyhalothrin, IMS e Immobilization stress, FSS e Forced swim stress.

the percentage of time spent in novel arm as compared to control.
3.2. Neurochemical studies
3.2.1. Effect on muscarinic-cholinergic receptors and mRNA
expression of CHRM2 receptor gene in frontal cortex and
hippocampus
Exposure to LCT and stress resulted to affect the binding of
cholinergic e muscarinic receptors in frontal cortex e LCT
(F1,12 ¼ 79.93, p < 0.001), stress (F2,12 ¼ 8.58, p < 0.01) and hip-
pocampus e LCT (F1,12 ¼ 69.51, p < 0.001), stress (F2,12 ¼ 15.46,
p < 0.001) on analyzing the data by two way ANOVA. A significant
interaction between LCT and stress exposure was observed in
hippocampus (F2,12 ¼ 6.31, p < 0.05) but not in frontal cortex
(F2,12 ¼ 0.74, p > 0.05). The regression analysis revealed that LCT
128 {95%C.I: 74e182, p < 0.001}, IMS 156 {95%C.I: 90e223,
p < 0.001} and FSS 139 {95%C.I: 72e205, p < 0.01} caused signifi-
cant decrease in the binding of cholinergic e muscarinic receptors
in frontal cortex as compared to control (Fig. 3A). Further, all
combinations of group were compared by one way ANOVA in
hippocampus. Exposure of rats to either LCT or IMS or FSS alone had
no significant effect on the binding of 3H-QNB to hippocampal
membranes, known to label cholinergic-muscarinic receptors as
compared to rats in the control group. A significant decrease in the
binding of 3H-QNB to hippocampal membranes (F(5,24) ¼ 18.39, 51%,
p < 0.001; 32%, p < 0.01) was evident in rats exposed to IMS or FSS
on LCT treatment as compared to rats in the control group (Fig. 4A).
Rats exposed to IMS or FSS on LCT treatment exhibited decrease in
the binding of 3H-QNB to hippocampal membranes (F(5,24) ¼ 18.39,
48%, p < 0.01; 27%, p < 0.01) as compared to those exposed to LCT
alone (Fig. 3A). Exposure of rats to IMS on LCT treatment also
caused decrease in the binding of 3H-QNB to hippocampal
(F(5,24) ¼ 18.39, 43%, p < 0.001) membranes as compared to those
subjected to IMS alone. Consistent with this, exposure of rats to FSS
on LCT treatment was also found to decrease the binding of
cholinergic e muscarinic receptors in hippocampal membranes
(F(5,24) ¼ 18.39, 20%, p < 0.05) in comparison to those subjected to
FSS alone. Scatchard analysis revealed that decrease in the binding
of cholinergic-muscarinic receptors on exposure of rats to IMS and
FSS on LCT treatment was due to decrease in the number of binding
sites (Bmax) and no change in the affinity (Kd) both in frontal
cortex and hippocampus as compared to rats exposed to LCT or IMS
or FSS alone (Table 1). The two-way ANOVA revealed that exposure
to LCT and stress modified the Bmax in frontal cortex e LCT
(F1,6 ¼ 17.94, p < 0.01), stress (F2,6 ¼ 8.58, p < 0.05) and hippo-
campus e LCT (F1,6 ¼ 39.57, p < 0.001), stress (F2,6 ¼ 6.45, p < 0.05)
however, there was no interaction between LCT and stress exposure
in frontal cortex (F2,6 ¼ 1.86, p < 0.05) and hippocampus
56 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63

A. 3H-QNB Binding

pmoles H-QNB bound/g protein

800 CONT LCT 700 CONT LCT
pmoles 3H-QNB bound/g protein

700 600
600
500
500 **b
***b 400
***a
400
300 ***a
300
200
200

3
100 100

0 0
No Stress IMS FSS No Stress IMS FSS
Frontal cortex Hippocampus

B. mRNA expression of CHRM2 receptor gene

mRNA level relative to control

mRNA level relative to control

1.2 CONT LCT 1.2 CONT LCT

1 1

0.8 0.8

0.6 *b 0.6 *b

0.4 **a 0.4 **a

0.2 0.2
0 0
No Stress IMS FSS No Stress IMS FSS
Frontal cortex Hippocampus
Fig. 3. Effect on 3H-QNB binding and mRNA expression of CHRM2 receptor gene in frontal cortex and hippocampus following repeated exposure of rats to stressors for 28 days and
lambda-cyhalothrin for 3 days. Values are mean ± SEM of five rats in 3H-QNB binding and three rats in mRNA expression in each group. The data was analyzed by two-way ANOVA
followed by Bonferroni test. LCT exposure decreased 3H-QNB binding and mRNA expression of CHRM2 receptor gene in frontal cortex and hippocampus in rats subjected to IMS or
FSS as compared to those exposed to IMS or FSS alone respectively. There was a significant interaction (p < 0.05) between LCT and stress on 3H-QNB binding in hippocampus only. a-
compared to IMS group, b-compared to FSS group. Significantly differs (*p < 0.05, **p < 0.01, ***p < 0.001). CONT e Control, LCT e Lambdaecyhalothrin, IMS e Immobilization
stress, FSS e Forced swim stress.

(F2,6 ¼ 2.45, p > 0.05) (Table 1). Interestingly, a significant decrease
in the Bmax of cholinergic-muscarinic receptors was observed on
regression analysis in frontal cortex e LCT 411 {95%C.I: 173e650,
p < 0.01}, IMS 424 {95%C.I: 132e716, p < 0.01} and FSS 373 {95%C.I:
81e665, p < 0.05} and hippocampus e LCT 418 {95%C.I: 226e611,
p < 0.001}, IMS 419 {95%C.I: 183e654, p < 0.001} and FSS 319 {95%
C.I: 83e554, p < 0.01} as compared to control.
The two-way ANOVA revealed that exposure to LCT and stress
affect the expression of CHRM2 receptor gene in frontal cortex e
LCT (F1,6 ¼ 46.41, p < 0.001), stress (F2,6 ¼ 7.18, p < 0.05) and hip-
pocampus e LCT (F1,6 ¼ 30.41, p < 0.01), stress (F2,6 ¼ 9.28, p < 0.05)
however, no interaction was observed between LCT and stress
exposure both in frontal cortex (F2,6 ¼ 2.55, p > 0.05) and hippo-
campus (F2,6 ¼ 2.27, p > 0.05). The regression analysis further
showed significant decrease in the expression of CHRM2 receptor
gene in frontal cortex e LCT 0.30 {95%C.I: 0.14e0.45, p < 0.001}, IMS
0.33 {95%C.I: 0.15e0.52, p < 0.01} and FSS 0.30 {95%C.I: 0.11e0.48,
p < 0.01} and hippocampus e LCT 0.33 {95%C.I: 0.18e0.49,
p < 0.001}, IMS 0.35 {95%C.I: 0.16e0.54, p < 0.001} and FSS 0.26
{95%C.I: 0.07e0.45, p < 0.01} as compared to rats in the control
group (Fig. 3B).
3.2.2. Effect on the expression of brain choline acetyltransferase and
mRNA expression of ChAT gene in frontal cortex and hippocampus
The two-way ANOVA exhibited that LCT and stress affect the
expression of ChAT in frontal cortex e LCT (F1,6 ¼ 15.54, p < 0.01),
stress (F2,6 ¼ 14.73, p < 0.01) and hippocampus e LCT (F1,6 ¼ 108,
p < 0.001), stress (F2,6 ¼ 18.73, p < 0.01) but there was no significant
interaction between LCT and stress exposure in frontal cortex
(F2,6 ¼ 2.63, p > 0.05) and hippocampus (F2,6 ¼ 2.27, p > 0.05). There
was significant decrease in the expression of ChAT in frontal cortex
e LCT 0.18 {95%C.I: 0.07e0.28, p < 0.01}, IMS 0.22 {95%C.I:
0.09e0.35, p < 0.01} and FSS 0.15 {95%C.I: 0.02e0.28, p < 0.05} and
hippocampus e LCT 0.42 {95%C.I: 0.26e0.57, p < 0.001}, IMS 0.45
 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63 57

A. Protein expression of choline acetyltransferase

1.2 CONT LCT 1.2 CONT LCT

Fold Change (% Control)

Fold Change (% Control)

1.0 1.0

0.8 *b 0.8
**a
0.6 0.6 **b
***a
0.4 0.4
0.2 0.2
0.0 0.0
No Stress IMS FSS No Stress IMS FSS
Frontal cortex Hippocampus

B. mRNA expression of choline acetyltransferase gene

mRNA level relative to control

1.2 CONT LCT 1.2 CONT LCT

mRNA level relative to control

1 1

0.8 0.8

0.6 *b
0.6 *b
*a *a
0.4 0.4

0.2 0.2

0 0
No Stress IM S FSS No Stress IMS FSS
Frontal cortex Hippocampus

Fig. 4. Effect on the expression of choline acetyltransferase protein and mRNA of choline acetyltransferase gene in frontal cortex and hippocampus following repeated exposure of
rats to stressors for 28 days and lambda-cyhalothrin for 3 days. Values are mean ± SEM of three rats in each group. The data was analyzed by two-way ANOVA followed by
Bonferroni test. LCT exposure decreased the expression of ChAT protein and mRNA of ChAT gene in frontal cortex and hippocampus in rats subjected to IMS or FSS as compared to
those exposed to IMS or FSS alone respectively. There was no significant interaction (p > 0.05) between stress and LCT on the expression of ChAT protein and mRNA of ChAT gene in
frontal cortex and hippocampus. a-compared to IMS group, b-compared to FSS group. Significantly differs (*p < 0.05, **p < 0.01, ***p < 0.001). CONT e Control, LCT e Lamb-
daecyhalothrin, IMS e Immobilization stress, FSS e Forced swim stress.

Table 1
Scatchard analysis of 3H-QNB binding in frontal cortex and hippocampus following repeated exposure of rats to stressors for 28 days and lambda-cyhalothrin for 3 days.

Type of stress Vehicle/LCT Frontal cortex Hippocampus

Kd Bmax Kd Bmax

No Stress CONT 1.37 ± 0.13 1313 ± 137 1.60 ± 0.11 1405 ± 112
LCT 1.19 ± 0.12 1262 ± 124 1.58 ± 0.16 1215 ± 103
IMS CONT 1.41 ± 0.19 1168 ± 131 1.60 ± 0.07 1171 ± 98
LCT 1.22 ± 0.12 557 ± 75*a 1.39 ± 0.11 609 ± 97**a
FSS CONT 1.35 ± 0.15 1201 ± 128 1.55 ± 0.16 1243 ± 131
LCT 1.26 ± 0.14 626 ± 85*b 1.31 ± 0.08 738 ± 69*b

Values are mean ± SEM of three rats in each group. The data was analyzed by two-way ANOVA followed by Bonferroni test. LCT exposure decreased the Bmax in frontal cortex
and hippocampus in rats subjected to IMS or FSS as compared to those exposed to IMS or FSS alone respectively.
Significantly differs (*p < 0.05, **p < 0.01). CONT e Control, LCT e Lambdaecyhalothrin, IMS e Immobilization stress, FSS e Forced swim stress.
a
Compared to immobilization stress.
b
Compared to forced swim stress.
58 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63
{95%C.I: 0.26e0.64, p < 0.001} and FSS 0.31 {95%C.I: 0.12e0.50,
p < 0.001} as compared to controls on regression analysis (Fig. 4A).
Exposure to LCT and stress also resulted to affect the mRNA
expression of ChAT gene in frontal cortex e LCT (F1,6 ¼ 31.18,
p < 0.01), stress (F2,6 ¼ 6.44, p < 0.05) and hippocampus e LCT
(F1,6 ¼ 28.04, p < 0.01), stress (F2,6 ¼ 11.51, p < 0.01) although no
interaction was observed both in frontal cortex (F2,6 ¼ 1.82,
p > 0.05) and hippocampus (F2,6 ¼ 6.90, p > 0.05) as revealed by
two way ANOVA (Fig. 4B). The regression analysis further exhibited
that there was a significant decrease in the expression of ChAT gene
in frontal cortex e LCT 0.30 {95%C.I: 0.16e0.45, p < 0.001}, IMS 0.29
{95%C.I: 0.11e0.47, p < 0.01} and FSS 0.25 {95%C.I: 0.07e0.43,
p < 0.01} and hippocampus e LCT 0.33 {95%C.I: 0.18e0.48,
p < 0.001}, IMS 0.35 {95%C.I: 0.16e0.53, p < 0.001} and FSS 0.29
{95%C.I: 0.11e0.48, p < 0.01} as compared to control.
3.2.3. Effect on activity of acetylcholinesterase and mRNA
expression of AChE gene in frontal cortex and hippocampus
Both LCT and stress were found to affect the activity of AChE in
frontal cortex e LCT (F1,12 ¼ 5.03, p < 0.001), stress (F2,12 ¼ 1.99,
p < 0.01) and hippocampus e LCT (F1,12 ¼ 7.21, p < 0.001), stress
(F2,12 ¼ 2.95, p < 0.05) while no interaction was observed between
LCT and stress exposure in frontal cortex (F2,12 ¼ 2.15, p > 0.05) and
hippocampus (F2,12 ¼ 1.49, p > 0.05) on two way ANOVA (Fig. 5A).
The regression analysis exhibited that there was a significant
decrease in AChE activity in frontal cortex e LCT 0.81 {95%C.I:
0.43e1.20, p < 0.001}, IMS 0.87 {95%C.I: 0.40e1.35, p < 0.001} and
FSS 0.58 {95%C.I: 0.10e1.05, p < 0.01} and hippocampus e LCT 0.98
{95%C.I: 0.47e1.49, p < 0.001}, IMS 1.04 {95%C.I: 0.42e1.67,
p < 0.001} and FSS 0.77 {95%C.I: 0.14e1.39, p < 0.01} as compared to
control.
It was further found that LCT and stress modified the mRNA
expression of AChE gene in frontal cortex e LCT (F1,6 ¼ 39.01,
p < 0.01), stress (F2,6 ¼ 8.22, p < 0.05) and hippocampus e LCT
(F1,6 ¼ 46.47, p < 0.001), stress (F2,6 ¼ 8.26, p < 0.05) although there
was no interaction in the mRNA expression of AChE gene between
them both in frontal cortex (F2,6 ¼ 1.99, p > 0.05) and hippocampus
(F2,6 ¼ 2.76, p > 0.05) on analyzing the data by two way ANOVA. The
regression analysis revealed that was a significant decrease in the
expression of AChE gene in frontal cortex e LCT 0.31 {95%C.I:
0.18e0.45, p < 0.001}, IMS 0.29 {95%C.I: 0.12e0.45, p < 0.001} and
FSS 0.27 {95%C.I: 0.11e0.44, p < 0.01} and hippocampus e LCT 0.32
{95%C.I: 0.17e0.48, p < 0.001}, IMS 0.34 {95%C.I: 0.15e0.52,
p < 0.001} and FSS 0.31 {95%C.I: 0.12e0.50, p < 0.01} as compared
to rats in the control group (Fig. 5B).
3.2.4. Effect on the expression of brain PKC b-1
Although, exposure to LCT and stress modified the expression of
PKC b-1 in frontal cortex e LCT (F1,6 ¼ 37.66, p < 0.001), stress
(F2,6 ¼ 5.32, p < 0.05) and hippocampus e LCT (F1,6 ¼ 92.67,
p < 0.001), stress (F2,6 ¼ 6.83, p < 0.05), no interaction between LCT
and stress exposure was evident both in frontal cortex (F2,6 ¼ 1.30,
p > 0.05) and hippocampus (F2,6 ¼ 1.56, p > 0.05) on analyzing the
data by two-way ANOVA (Fig. 6). A significant decrease in the
expression of PKC b-1 was observed both in frontal cortex e LCT
0.27 {95%C.I: 0.15e0.39, p < 0.001}, IMS 0.21 {95%C.I: 0.06e0.35,
p < 0.01} and FSS 0.21 {95%C.I: 0.06e0.35, p < 0.01} and hippo-
campus e LCT 0.33 {95%C.I: 0.17e0.48, p < 0.001}, IMS 0.37 {95%C.I:
0.18e0.55, p < 0.001} and FSS 0.34 {95%C.I: 0.15e0.53, p < 0.01} on
carrying out the regression analysis as compared to control.
4. Discussion
Although neurotoxicity of pyrethroids is largely attributed to
their effects on the gating characteristics of voltage sensitive
sodium and calcium channels, a number of experimental studies
have found that pyrethroid exposure can also affect the functioning
of the brain neurotransmitter system in insects, mammals and
other species (Ray and Fry, 2006; Righi and Palermo-Neto, 2003;
Soderlund, 2012). Recently, Li et al. (2014) found tissue specific
damage in gold fish (Carassius auratus) by sub-lethal exposure to
LCT using an NMR based metabolomics approach. Exposure to LCT
resulted to enhance glutamate and decrease glutamine levels
associated with a slight increase in GABA levels in the brain, sug-
gesting that the glutamine-glutamate-GABA axis could play an
important role in LCT induced neurotoxicity. In view of vulnera-
bility of brain dopaminergic and cholinergic systems, studies were
carried out by us earlier to assess the impact of LCT on developing
rats to understand whether behavioral and neurochemical changes
following LCT exposure are transient or persistent (Ansari et al.,
2012a,b). Repeated exposure to LCT (1 or 3 mg/kg body weight)
in weanling rats from the postnatal day (PD) 22 to PD49 resulted to
decrease the binding of brain muscarinic-cholinergic receptors
associated with decrease in AChE activity and impaired learning
and memory (Ansari et al., 2012a). The behavioral and neuro-
chemical changes were more marked in rats exposed to LCT at a
dose of 3 mg/kg body weight and continued to persist even on
withdrawal of LCT exposure on PD65 (Ansari et al., 2012a). In the
present study, the dose 3 mg/kg body weight of LCT was based on
the pilot experiments carried out by us to assess the non-toxic dose
that would not have effect on the key behavioral and neurochem-
ical parameters associated with brain cholinergic system. Rats were
treated at three different doses (1, 3 or 7 mg/kg body weight, p.o)
for 3 days and the effect on the passive avoidance response, fore
limb grip strength and brain cholinergic-muscarinic receptors was
assessed 24 h after the last dose of LCT. Treatment with LCT (7 mg/
kg body weight, p.o) for 3 days resulted to decrease the binding of
cholinergic-muscarinic receptors both in frontal cortex and hip-
pocampus associated with impairment in learning and muscle
strength. However, no significant change in any of the behavioral
and neurochemical endpoint was observed in rats treated either at
1 or 3 mg/kg body weight dose of LCT for 3 days in the pilot study. In
the present study, there was no significant effect on the binding of
muscarinic-cholinergic receptors, CHRM2 receptor gene, AChE ac-
tivity and AChE gene in the frontal cortex and hippocampus of rats
on exposure to LCT (3 mg/kg body weight) for 3 days (26th, 27th
and 28th day of treatment). Consistent with this, no significant
effect on the expression of ChAT mRNA and protein in both brain
regions and learning and memory was observed on LCT exposure
(3 mg/kg body weight) for 3 days (26th, 27th and 28th day of
treatment) in these rats. In another study by us, no significant
change in brain biogenic amines and BBB permeability was
observed in rats treated with LCT (3 mg/kg body weight) for 3 days
(Shukla et al., 2015). Earlier study was carried out on female
weanling rats and exposure to LCT (1 or 3 mg/kg body weight, p.o)
was for 28 days during early life, however, in the present study,
adult male rats have been used and the duration of LCT treatment
(3 mg/kg body weight, p.o) was for 3 days. Therefore, no significant
changes in brain cholinergic parameters in the present study may
be attributed to age and gender which are important contributors
in the toxicity of chemicals. While investigating the behavioral ef-
fects of cyhalothrin Righi and Palermo-Neto (2003) found that
treatment with commercial formulation of cyhalothrin caused
anxiety like symptoms associated with increased serum cortios-
terone levels in rats treated with LCT at 3 or 7 mg/kg body weight
dose and not in those treated at 1 mg/kg body weight dose for 7
days.
Although modifications in brain cholinergic system in response
to stress has been reported by a number of investigators, consistent
changes have not been observed (Abdel-Rahman et al., 2004;
 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63 59

A. Acetylcholinesterase activity
μmole hydrolyzed / min/ mg protien
4 CONT LCT 5 CONT LCT

μmole hydrolyzed / min/ mg protien

4
3
*b 3
**b
2 **a **a
2

1 1

0 0
No Stress IMS FSS No Stress IMS FSS
Frontal cortex Hippocampus

B. mRNA expression of acetylcholinesterase activity gene

1.2 CONT LCT
mRNA level relative to control
mRNA level relative to control

1.2 CONT LCT

1
1
0.8
0.8
0.6
0.6 *b **b
**a 0.4 **a
0.4
0.2 0.2

0 0
No Stress IMS FSS No Stress IMS FSS
Hippocampus
Frontal cortex
Fig. 5. Effect on acetylcholinesterase activity and mRNA expression of acetylcholinesterase gene in frontal cortex and hippocampus following repeated exposure of rats to stressors
for 28 days and lambda-cyhalothrin for 3 days. Values are mean ± SEM of five rats incase of AChE activity and three rats in case of mRNA expression in each group. The data was
analyzed by two-way ANOVA followed by Bonferroni test. LCT exposure decreased the AChE activity and mRNA expression of AChE gene in frontal cortex and hippocampus in rats
subjected to IMS or FSS as compared to those exposed to IMS or FSS alone respectively There was no significant interaction (p > 0.05) between stress and LCT on the acetyl-
cholinesterase activity and mRNA expression of AChE gene in both the brain regions. Significantly differs (*p < 0.05, **p < 0.01). CONT e Control, LCT e Lambdaecyhalothrin, IMS e
Immobilization stress, FSS e Forced swim stress.

Gonz alez and Pazos, 1992a,b; Song et al., 2002). Type of stressor(s),
frequency and duration of stress appear to be important de-
terminants that could affect the integrity of brain cholinergic sys-
tem including cholinergic receptors. Regional response of brain
cholinergic receptors was studied in rats subjected to IMS for
different time periods (Takayama et al., 1987). Increase in the
binding of cholinergic receptors was evident in hippocampus,
striatum, septum and pons-medulla of rats subjected to IMS for 0.5,
1 or 3 h. Acute IMS for 2 h resulted to increase the affinity of 3HeN-
methylscopolamine (NMS), known to label cholinergic-muscarinic
receptors and not in those subjected to IMS for 10 min (Gonz alez
and Pazos, 1992a). In another study, increase in the cholinergic-
muscarinic receptors was observed in the brain of rats subjected
to IMS for 2 h daily for 3, 7 or 21 days and not in those exposed to
IMS for 10 min daily for 3, 7 or 21 days (Gonzalez and Pazos, 1992b).
In the pilot studies carried out by us to assess the effect of IMS or
FSS on brain cholinergic-muscarinic receptors, rats were subjected
to IMS (15, 30 or 60 min/day) or FSS (3, 5 or 15 min/day) for 28 days.
No significant change in the binding of cholinergic-muscarinic re-
ceptors was observed in rats subjected to IMS for 15 min/day or FSS
for 3 min/day for 28 days suggesting that stress for a short period
may not affect the sensitivity of brain cholinergic-muscarinic
receptors.
While investigating the effect of concurrent exposure to
repeated stress and chlorpyrifos, a significant decrease in the
binding of cholinergic-muscarinic receptors was observed due to
decreased Bmax in rats subjected to restraint (1 h/day; 5 days/
week) or swim (30 min/day; 1 day/week) stress for 28 days on
chlorpyrifos exposure in cerebral cortex and not in hippocampus
(Pung et al., 2006). No significant change in the binding of
cholinergic-muscarinic receptors was observed in hippocampus
and cerebral cortex of rats subjected to either restraint or
60 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63

1.2 CONT LCT 1.2 CONT LCT

Fold Change (% Control)

Fold Change (% Control)

1.0 1.0

0.8 0.8
*b
**a
0.6 0.6 *b
***a
0.4 0.4
0.2 0.2
0.0 0.0
No Stress IMS FSS No Stress IMS FSS
Frontal cortex Hippocampus
Fig. 6. Effect on the expression of PKC b-1 protein following repeated exposure of rats to stressors for 28 days and lambda-cyhalothrin for 3 days. Values are mean ± SEM of three
rats in each group. The data was analyzed by two-way ANOVA followed by Bonferroni test. LCT exposure decreased the expression of PKC b-1 protein in frontal cortex and hip-
pocampus in rats subjected to IMS or FSS as compared to those exposed to IMS or FSS alone respectively. There was no significant interaction (p > 0.05) between stress and LCT on
the expression of PKC b-1 protein in both the brain regions. a-compared to immobilization stress, b-compared to forced swim stress. Significantly differs (*p < 0.05, **p < 0.01,
***p < 0.001). CONT e Control, LCT e Lambda-cyhalothrin, IMS e Immobilization stress, FSS e Forced swim stress.

swimming (30 min/day; 1 day/week) stress for 28 days (Pung et al.,
2006). While studying the influence of stress on the neurotoxicity
of chemicals used in the gulf war (pyridostigmine bromide, DEET
and permethrin), decrease in the binding of M2 cholinergic-
muscarinic receptors was observed in the brain stem and not in
mid brain and cerebellum of rats subjected to restraint stress for
5 min/day for 28 days as compared to controls. Rats subjected to
restraint stress on co-exposure to these chemicals, however,
resulted to decrease the binding of M2 cholinergic-muscarinic re-
ceptors in mid brain and cerebellum (Abdel-Rahman et al., 2004).
In another study, decrease in the binding of cholinergic-muscarinic
receptors in the fore brain was observed in rats subjected to re-
straint stress for 5 min on co-exposure to these chemicals (Abdel-
Rahman et al., 2002). In the present study, pre-exposure to IMS
or FSS followed by LCT treatment resulted to decrease the binding
of cholinergic-muscarinic receptors in frontal cortex and hippo-
campus. Decrease in the expression of CHRM2 receptor gene both
in frontal cortex and hippocampus was also observed in these rats
suggesting vulnerability of brain cholinergic system to LCT under
stress.
A number of studies have found that changes in post-synaptic
cholinergic-muscarinic receptors could be associated with alter-
ations in the activity of AChE, an enzyme involved in the meta-
bolism of acetylcholine, a principal neurotransmitter involved in
cholinergic neurotransmission (Ansari et al., 2012a; Chandravanshi
et al., 2014; Lee et al., 2009). Besides, integrity of pre-synaptic
events, specially the activity of ChAT, an enzyme involved in the
synthesis of acetylcholine and a marker for the integrity of
cholinergic neurons is also important and any fluctuation in its
activity could affect the sensitivity of brain cholinergic-muscarinic
receptors. It has been found that stress could affect the activity of
brain ChAT and AChE of the fact that stressors, their intensity and
duration of exposure may be different (Oh et al., 2007; Rao and
Raju, 2000, Sunanda et al., 2000; Gottesfeld et al., 1978; Tsakiris
et al., 2008). Earlier, stress had been found to decrease acetylcho-
line levels (Anisman et al., 1981). Involvement of AChE in pyrethroid
neurotoxicity has been observed in different animal species.
Exposure to LCT resulted to decrease AChE activity in brain, gills
and muscles of Channa punctatus (Kumar et al., 2009). While
studying the neurotoxic effect of lamda-cyhalothrin, Piner and
Üner (2014) also observed that LCT and LCTe piperonyl butoxide
could inhibit AChE activity in the brain of Oreochromis niloticus by
modulating cytochrome P450 with piperonyl butoxide. Exposure of
weaned rats to LCT (1.0 or 3.0 mg/kg body weight) for 28 days
resulted to decrease the protein expression of ChAT and activity of
AChE in the brain and these changes were found to be associated
with decreased binding of cholinergic-muscarinic receptors in
brain regions in an earlier study by us (Ansari et al., 2012a). How-
ever, no change in protein expression of ChAT and activity of AChE
in frontal cortex and hippocampus on LCT exposure suggests that
LCT at this dose and duration has no effect on cholinergic system.
Interestingly, decrease in the expression of ChAT, a marker of
cholinergic neurons associated with decrease in the expression of
mRNA and AChE activity both in frontal cortex and hippocampus of
rats preexposed to IMS or FSS on LCT exposure indicate disruption
of the cholinergic system as observed in the present study. Further,
decrease in the expression of ChAT and AChE activity could increase
levels of acetylcholine and contribute to desensitization of
cholinergic-muscarinic receptors.
In a series of experiments carried out to assess the effect of
stressors on the toxicity of pyridostigmine, an anticholinergic
agent, no significant effect of restraint or forced swimming or
forced running stress was observed on the acute and sub-acute
toxicity of pyridostigmine (Shaikh and Pope, 2003; Song et al.,
2002; Tian et al., 2002). In contrast, FSS resulted to increase the
potential of pyridostigmine bromide in inhibiting the AChE activity
 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63
in mice brain and these changes were associated with disruption of
the BBB permeability (Friedman et al., 1996). Consistent with this,
neuronal injury was also observed on combined exposure of rats to
restraint stress and low doses of warfare agents including pyrido-
stigmine bromide due to disruption in the BBB permeability.
Further, these changes were linked with modifications in the brain
cholinergic functions including decreased AChE activity (Abdel-
Rahman et al., 2002). It has been suggested that disruption in the
BBB permeability increase the cerebrovascular permeability to the
chemicals/drugs due to increase in blood flow (Abou-Donia, 1996).
As a result, the compounds which do not easily cross the blood
brain barrier could reach the brain and cause adverse effects.
Studies carried out earlier found that plasma corticosterone levels
could modulate the neurotoxicity of chlorpyrifos, an organophos-
phate pesticide (Pung et al., 2006). While there was a transient
change in plasma corticosterone levels that could remain high up to
40 min (Pung et al., 2006) or 5 h (Mizoguchi et al., 2001), enhanced
corticosterone levels could also be one of the important contribu-
tors for interaction with toxicants neurobehavioral changes
specially cholinergic functions. Righi and Palermo-Neto (2003) in
an interesting study also found a link of neurobehavioral alterations
with enhanced plasma corticosterone levels on repeated exposure
to cyhalothrin in rats. In a recent study, we also found that pre-
exposure of rats to IMS or FSS on LCT treatment increased the
BBB permeability and enhanced corticosterone levels but these
changes were not observed in rats exposed to IMS or FSS or LCT
alone (Shukla et al., 2015). Thus disruption in the BBB permeability
could be one of the possible reasons for cholinergic deficits in rats
pre-exposed to IMS or FSS on LCT treatment as observed in the
present study. Although the biochemical events associated with the
disruption of BBB permeability are not clearly understood, the
involvement of c-fos, an early response gene, enhanced glucocor-
ticoid and catecholamine levels and increased release of histamine,
a vasoactive mediator has been suggested convincingly (Friedman
et al., 1996; Melia et al., 1994; Esposito et al., 2001). Further stress
induced response on the BBB permeability has been found to vary
with age, species and strain of animals (Sharma and Dey, 1986;
Abdel-Rahman et al., 2002; Esposito et al., 2001; Colomina et al.,
2005). A number of studies have revealed that malnutrition may
significantly contribute to alter the HPA axis (Lesage et al., 2002,
2006; Xiong and Zhang, 2013) and thus the response of environ-
mental chemicals could be enhanced and implicated in the etiology
of a number of diseases including neurotoxicity. Human exposure
due to uncontrolled usage of pyrethroid including LCT even at low
dose may not be ruled out and could enhance toxicity under these
situations.
Impairment in learning and memory has been found on expo-
sure to stressor(s) or environmental chemicals including pyre-
throids (Abdel-Rahman et al., 2004; Cory-Slechta et al., 2010; Pung
et al., 2006; Nasuti et al., 2013; Habr et al., 2014). Both IMS and FSS
have been found to cause deficits in learning and memory in rats
assessed using different paradigms (Trofimiuk and Braszko, 2008).
Earlier, we found that repeated exposure of weaned rats to LCT (1.0
or 3.0 mg/kg body weight) affected learning and memory (Ansari
et al., 2012a). In the present study, no significant change in
learning and memory was observed in rats exposed to LCT (3.0 mg/
kg body weight) for 3 days or those subjected to IMS (15 min/day)
or FSS (3 min/day) stress for 28 days. However, pre-exposure to IMS
or FSS on LCT treatment resulted to affect the novelty seeking
behavior in rats, assessed by Y-maze and learning and memory
assessed by passive avoidance response. As cholinergic alterations
in hippocampus may affect the learning and memory (Conrad,
2010; Sapolsky, 2000; Wang et al., 2009), decreased cholinergic
activity in hippocampus and frontal cortex of rats pre-exposed to
IMS or FSS on LCT exposure as observed in the present study could
61
be associated with impairment in learning and memory. Role of
PKC b-1, a signaling protein in the brain has been suggested in
modulating long term potentiation and associated with learning
and memory (Paratcha et al., 2000; Wu et al., 2007). Interestingly,
decreased expression of PKC b-1 in hippocampus of rats pre-
exposed to either of the stressors followed by LCT treatment
could be associated with impaired learning and memory as
observed by us earlier on exposure to arsenic (Chandravanshi et al.,
2014). Further, it has been found that prolonged stress could
enhance corticosterone levels, disrupt brain cholinergic neurons
and impair spatial memory (Conrad, 2010; Luine and Rodriguez,
1994; McEwen and Sapolsky, 1995). Enhanced plasma corticoste-
rone levels were observed in a recent study by us on pre-exposure
of rats to IMS or FSS followed by LCT treatment and not in those
exposed to either of the stressors or LCT alone (Shukla et al., 2015).
The results of the present study exhibit that pre-exposure of rats
to IMS or FSS on LCT treatment could significantly contribute in
brain cholinergic dysfunctions associated with impaired learning
and memory (Fig. 7). Further, neurobehavioral changes appear to be
more intense in rats subjected to IMS as compared to those exposed
to FSS. The findings in the present study appear to be interesting as
the changes were not observed in rats exposed to the stressors or
Immobilization Forced swim
stress stress
(15 min / day) Or (3 min / day)
for 28 days for 28 days
Lambda- cyhalothrin
exposure
(3.0 mg/kg bw, p.o) on
days 26, 27 and 28
ChAT, AChE
CHRM Receptors
PKC β-1
in Frontal cortex and Hippocampus
Brain Cholinergic Deficits
Impaired learning & memory and
muscle strength
Fig. 7. Brain Cholinergic dysfunctions in rats pre-exposed to immobilization or forced
swim stress on lambda-cyhalothrin treatment. Rats subjected to either IMS (15 min/
day for 28 days) or FSS (3 min/day for 28 days) or treated with LCT (3.0 mg/kg, body
weight, p.o, for 3 days) alone had marginal changes on brain cholinergic functions. Pre-
exposure of rats either to IMS or FSS for 28 days on LCT treatment for 3 days resulted to
affect behavioral and neurochemical endpoints associated with brain cholinergic
system.
62 R.K. Shukla et al. / Neurochemistry International 93 (2016) 51e63
LCT alone. Further studies are required to assess the impact on
other neurotransmitter systems.
Acknowledgment
The authors thank Director, CSIR e Indian Institute of Toxicology
Research (CSIR-IITR), Lucknow for his keen interest in the present
study. The study is part of the INDEPTH programme of Council of
Scientific and Industrial Research, New Delhi. CSIReIITR, Lucknow
Communication no. 3323. The support from Indian Council of
Medical Research, New Delhi for the award of Senior Research
Fellowship to Rajendra K. Shukla is acknowledged. The authors also
thank Professor (Ms). Madhu Mehrotra, Former Head, Department
of English and Eurpean Languages, University of Lucknow, Lucknow
in editing the manuscript. The authors also acknowledge the help of
Mr. B.S. Pandey for extending the technical assistance.
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