Physiology & Behavior 156 (2016) 148–155

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Physiology & Behavior

journal homepage: www.elsevier.com/locate/phb

Memory and learning seems to be related to cholinergic dysfunction in

the JE rat model
Prashant Singh Chauhan a, Usha Kant Misra a,⁎, Jayantee Kalita a,
Lalit Pratap Chandravanshi b, Vinay Kumar Khanna b
a
Department of Neurology, Sanjay Gandhi Postgraduate Institute of Medical Science, Raebareily Road, Lucknow, India
b
Developmental Toxicology Division, CSIR-Indian Institute of Toxicology Research, Post Box 80, MG Marg, Lucknow, India

H I G H L I G H T S

• Intracerebral inoculation of JEV (3 × 106 pfu/ml) on 12 day Wistar rats.

• Spatial memory and learning was impaired on 10 and 33 dpi and recovered on 48 dpi.
• Cholinergic markers were reduced in cortex, hippocampus, striatum, and cerebellum.
• Correlation between spatial memory and brain cholinergic parameters was found.
• Transient form of spatial memory impairment was observed in JE infected rats.

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

Article history: Cognitive changes have been known in encephalitis but in Japanese encephalitis (JE) such studies are limited.
Received 13 June 2015 This study aims at evaluating the spatial memory and learning and correlate with markers of cholinergic activity
Received in revised form 12 October 2015 in the brain.12 day old Wistar rats were inoculated with dose of 3 × 106 pfu/ml of JE virus. On 10, 33 and 48 days
Accepted 9 January 2016
post-inoculation (dpi), spatial memory and learning was assessed by Y maze. Brain biopsies from frontal cortex,
Available online 11 January 2016
corpus striatum, hippocampus and cerebellum were taken. Muscarinic cholinergic receptor was assayed by
Keywords:
Quinuclidinyl benzylate (H3-QNB) binding, CHRM2 gene expression by real time PCR and choline acetyl transfer-
Memory ase (ChAT) by Western blot. Spatial learning and memory showed significant decline in rats inoculated with JEV
Learning on 10 and 33 dpi (47.5%, p b 0.01; 30.2%, p b 0.01). It started recovering on 48 dpi. Muscarinic cholinergic recep-
Viral encephalitis tors showed significant decrease in frontal cortex (31%, p = 0.001; 26%, p = 0.003), hippocampus (57%, p =
Muscarinic receptor 0.001; 39.9%, p = 0.002) and cerebellum (31.2%, p = 0.008; 21.6%, p = 0.007) but not in corpus striatum as
Cognition compared to control. The mRNA expression of CHRM2 receptor gene showed significant decrease in the expres-
Brain regions sion in frontal cortex (48%, p b 0.001; 38%, p b 0.01), hippocampus (43%, p b 0.001; 37%, p b 0.05) and cerebellum
(46%, p b 0.001; 42%, p b 0.05) on 10 and 33 dpi. ChAT showed significant fold decrease in the expression in fron-
tal cortex (2.11, p b 0.01, 1.12, p b 0.05) and hippocampus (2.2, p b 0.01, 1.41, p b 0.05) on 10 and 33 dpi. Corre-
lation between ChAT, CHRM2 and total muscarinic receptor activity with spatial memory were found at different
dpi. There was transient spatial learning and memory impairment which was associated with reduction of total
muscarinic receptor binding, CHRM2 gene and ChAT expression in different brain region of rat infected with JE
Virus.
© 2016 Elsevier Inc. All rights reserved.

1. Introduction
Japanese encephalitis is one of the commonest causes of infectious
encephalitis in the world and affects nearly 70,000 cases with 25–40%
annual mortality. Nearly half of survivors of JE develop neurological
sequelae [14,21,24,31]. Cognitive and behavioral changes constitute
⁎ Corresponding author at: Department of Neurology, Sanjay Gandhi Post Graduate
Institute of Medical Sciences, Raebareily Road, Lucknow 226014, India.
E-mail addresses: drukmisra@rediffmail.com, ukmisra@sgpgi.ac.in (U.K. Misra).
about 20–30% of the sequelae [47,50]. JE affects specific areas of brain
as has been shown in MRI and autopsy studies. MRI study in six patients
with JE revealed bilateral thalamic involvement in five, brainstem in
three and basal ganglia in one [32]. Signal changes were also seen in
midbrain and cerebellum in three each of seven patients and the basal
ganglia in one [25] and another study on seventeen patients also re-
vealed similar findings [20]. MR imaging in 62 patients with JE revealed
temporal lobe (mostly hippocampus) involvement in 11 (17.7%) pa-
tients in addition to thalamic and brainstem involvement [16].These
changes are consistent with human autopsy studies in which the lesions

http://dx.doi.org/10.1016/j.physbeh.2016.01.006
0031-9384/© 2016 Elsevier Inc. All rights reserved.
 P.S. Chauhan et al. / Physiology & Behavior 156 (2016) 148–155
were noted in thalamus, basal ganglia, midbrain, cerebral cortex and
cerebellum [44]. The histopathological findings in 34 autopsy cases
form Karnataka India revealed edema, hazy meninges, focal congestion
in white matter and deep gray matter along with necrosis especially in
thalamus, corpus striatum, midbrain, pons and cerebellum [44]. These
results were in agreement with that of 11 autopsies from Japan [53].
Cholinergic neurons are present in the basal forebrain complex,
frontal cortex, striatum, hippocampus, amygdala and brainstem [4],
these region are also involved in JE. Cholinergic projections from basal
forebrain (nucleus basalis of Meynert) to the frontal cortex and limbic
structures (hippocampus) are associated with learning and memory
[30]. Cholinergic dysfunction has been linked to neurobehavioral
alterations in various neurological disorders such as Alzheimer's disease
(AD) [2], Huntington disease [46] schizophrenia [41] and, organophos-
phate poisoning (OP) [6].
Choline acetyl transferase (ChAT) is a sensitive indicator of choliner-
gic activity in the central nervous system (CNS). In AD, ChAT activity is
reduced by 90% in nBM (basal forebrain), along with other sub-cortical
areas such as hypothalamus, caudate, and thalamus but is normal in stri-
atum [1]. Loss of about 90% AChE activity has been reported in different
cortical areas in AD [36]. There is also decrease of up to 30% in the density
of muscarinic receptors predominantly in the hippocampus [36,37]. Out
of five muscarinic receptors (M1-M5), loss of M2 subtype is the most
prevalent in AD [28].
Brain cholinergic activity can be evaluated in different brain regions
of experimental animal and these can be correlated with memory and
learning abnormalities. Such studies may provide valuable information
on the possible mechanism of CNS infection on memory and behavioral
changes. There is paucity of studies evaluating the role of cholinergic
functions in CNS infections.
We hypothesize that JE may result in, memory and learning impair-
ment which will partially or completely recover on follow up as the
infection subsides. The memory and learning functions may correlate
with cholinergic activity in brain.
We aim to study the memory and learning in JEV infected rats at
different time points after JEV infection and to correlate these with cho-
linergic activity in the brain. We also study these changes at different
time points to study the recovery of cognitive and biochemical markers.
2. Material and methods
2.1. Virus
Indian neuro-virulent strain of JE virus, GP 78668A (GP-78) was
used for the viral titration by standard plaque assay [52]. To increase
the viral titer, intracerebral inoculation with 50 μl, 2% MEM (Gibco,
NY, USA) of JEV was injected in 3–4 days old suckling mice.
2.2. Animals
In the present study 12 days old suckling Wistar rats were used. The
rats were maintained under hygienic conditions in an air conditioned
room at 25 ± 2 °C with 12 h light/dark cycle. The rats were housed
with free access to food and water. The experimental protocol was
approved by the institutional Animal Ethics Committee. All the experi-
mental procedures were carried out in accordance with the institutional
guidelines.
2.3. Rat model of JE
120 rats were divided into two groups viz. Group 1 and Group 2. The
details of experimental design are presented in Fig. 1. JEV (3 × 106 pfu/ml)
was inoculated with minimum penetration of 5 mm into the cerebral
cortex, taking care to avoid injury in any specific brain region [39].
3 × 106 pfu virus dose of GP 78668A (GP-78) was used for intracerebral
inoculation because of the greater susceptibility and longer survival of
149
younger rats compared to the older ones. The viral dose used in this
study (3 × 106 pfu) was standardized on the basis of previous report
[23,39,48].
Sterile phosphate buffered saline (PBS) was inoculated in the control
rats following the same protocol. Both JEV inoculated and control
PBS inoculated rats were monitored daily for any clinical signs and
symptoms.
3. Behavioral parameters
3.1. Spatial learning and memory
3.1.1. Continuous alternation test
Spontaneous alternation in a single session was assessed in a
computerized Y maze (TSE, Germany), which was used as a measure
of short-term memory performance [29,43]. Each arm of Y maze was
40 cm long, 30 cm high and 15 cm wide and converged in an equilateral
triangular central area with 15 cm at its longest axis. Rat was placed at
the end of one arm and allowed to move freely through the maze for
five minutes. The sequence of each arm entry recorded automatically
by Y maze software. Measure of spatial memory was defined as the
number and the sequence of the arm entries were recorded during
5 min. The alternation percentage was calculated as the number of alter-
nations (entries into three different arms consecutively) divided by the
total possible alternations (i.e. the number of arms entered minus 2).
3.1.2. Neurotransmitter receptor binding assay
Cholinergic muscarinic receptors were estimated in frontal cortex,
hippocampus, corpus striatum and cerebellum, using radioligand recep-
tor binding technique following the standard procedure [22].
3.1.3. Preparation of crude synaptic membrane
Crude synaptic membrane was prepared by homogenizing the brain
regions (frontal cortex, corpus striatum, hippocampus and cerebellum)
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. The pellet was washed twice
in homogenization buffer and centrifuged at 40,000 g for 15 min at 4 °C.
The pellet was suspended in Tris–HCl buffer (40 mM, pH 7.4) and stored
at −20 °C for binding assays.
3.1.4. Assay of muscarinic–cholinergic receptors in selected brain regions
Binding incubations in a final volume of 1.0 ml were carried out in
triplicate. For muscarinic–cholinergic receptors, 3H-QNB (42 Ci/mmol,
Perkin Elmer, USA) was used and atropine sulfate (1.9 × 10−6 M) was
used as a competitor (Table 1). The reaction mixture containing Tris–
HCl buffer (40 mM, pH 7.4), together with membrane protein
(300–400 μg) and appropriate radioligand was incubated for 15 min at
37 °C in the presence or absence of competitor to assess the non-
specific and total binding respectively. At the end of incubation, contents
of the binding tubes were immediately filtered on glass fiber disks
(25 mm diameter, 0.3 μm pore size, Whatman GF/B) and washed
twice rapidly with 5 ml chilled Tris–HCl buffer (40 mM, pH 7.4). Filters
were dried and transferred into vials and scintillation mixture (2,5-
diphenyl oxazole; 1,4-bis-5, phenylozazolyl-benzene; naphthalene; tol-
uene; methanol and 1,4 dioxane) added to it. The radioactivity was
counted on β scintillation counter (Packard, USA) at an efficiency of
30–40% for 3H to determine membrane bound radioactivity. Specific
binding was determined by subtracting the non-specific binding from
the total binding and has been expressed as pmol ligand bound/g pro-
tein. The amount of membrane protein per tube was around 300–
350 mg as determined by the method [27].
3.1.5. Total RNA extraction
RNA was extracted from separated brain regions (frontal cortex, cor-
pus striatum, hippocampus and cerebellum) individually collected in
1 ml of ice-cold TRIzol (Invitrogen, Carlsbad, CA, USA) using QIAmp
150 P.S. Chauhan et al. / Physiology & Behavior 156 (2016) 148–155
Fig. 1. Distribution of rats for JEV inoculated (experimental group) and PBS inoculated (control group) on 10, 33 and 48 dpi. dpi = days post-inoculation, JEV = Japanese encephalitis virus.
RNA kit (QIAGEN, Inc., Valencia, California). RNA was eluted in 50 μl of
di-ethyl pyro carbonate (DEPC) treated distilled water and stored at
−80 °C till further use. The amount of RNA in each sample was quanti-
fied by measuring the absorbance at 260 nm using a spectrophotometer
(Hitachi, Japan).
3.1.6. cDNA synthesis
Single strand cDNA was synthesized from total RNA using the high
capacity cDNA reverse transcription kit (Applied Biosystems, USA), 2×
transcription master mix was prepared and for 10 μl reaction, 10× RT
buffer, 25× DNTP mix (100 mM), 10× RT random primer, multiscribe
reverse transcriptase and nucleus free water was added and kept in
ice. 10 μl of 2× RT master mix was pipetted into each individual tube.
10 μl of RNA sample was added into each tube and the tubes were
sealed. The tube was placed into thermal cycler using the conditions
such as step I 25 °C for 10 min, step II 37 °C for 120 min, step III 85 °C
for 5 min and step IV infinite.
3.1.7. Quantitative RT–PCR analysis
For quantitative PCR, cDNA synthesized by High-Capacity cDNA
Reverse Transcription Kit (Applied Biosystems, USA) was used. The se-
quence of primers used for CHRM2 and GAPDH [26]. The PCR reaction
mixture for CHRM2 and GAPDH in 20 μl contained 1× TaqMan Univer-
sal PCR Master Mix (Applied Biosystems), 10 pM of each gene primers,
2 μl cDNA and nuclease-free water. RT-PCR assay for each gene target
was performed in triplicate. The amplification was carried out using
the Roche 480 real time PCR system (USA). PCR conditions were 50 °C
for 2 min, 90 °C for 10 min, 95 °C for 15 s and 60 °C for 1 min. The
results have been analyzed by the 2−ΔΔCT method using GAPDH as a
reference gene [42].
3.1.8. Western blot quantification of protein expression
Expression of choline acetyl transferase (ChAT) protein in the frontal
cortex and hippocampus was carried out [19]. Fresh frozen brain
Table 1
Details of ligand, competitor used for receptor binding assay in different brain regions.
Receptor Brain region
Cholinergic–muscarinic Frontal cortex, corpus stratum, cerebellum, hippocampus
regions were homogenized by sonication in ice in RIPA buffer contain-
ing 50 mM Tris–HCl, pH 6.8, 150 nm NaCl, 0.5% sodium deoxycholate,
0.1% SDS, protease inhibitor, and 1% triton-100X with protein inhibitor
and centrifuged at 12,000 g for 15 min at 4 °C to remove the insoluble
material. The pellets were discarded and supernatant was further
mixed with loading buffer containing 60 mM Tris–HCl, pH 6.8, 2% w/v
SDS, 10% v/v glycerol, 5% b-mercaptoethanol, 0.01% w/v bromophenol
blue and boiled at 95 °C for 5–7 min. The prepared samples (30 μg pro-
tein/lane) were electrophoresed on 12% SDS-PAGE, before transfer to
hybond nitrocellulose membranes (Millipore, USA) and blocked with
blocking buffer (Western blocker solution, TM, Sigma, USA). After
washing, the membrane was incubated with primary antibody (Anti-
ChAT, 1:2000, Sigma, USA) for 24 h at 4 °C followed by incubation
with horseradish peroxidase linked secondary antibody (anti-mouse
IgG, 1:4000) at room temperature for 60 min. After the incubation,
blots were washed and developed using a based detection was per-
formed with Immobilon Western chemiluminescent HRP substrate
(Millipore, USA) following the recommended procedure. To normalize
the protein bands to gel loading control, β actin was probed as an inter-
nal control and used to confirm that an equal amount of protein was
loaded in each lane. A digital gel image analysis system (VersaDoc,
Model 1000, Bio Rad, Quantity 1) was used for semi-quantification of
ChAT immunoreactivity.
3.1.9. Statistical Analysis
Data generated from behavior analysis, Western blot and receptor
binding assay were analyzed by Student's t test or one way analysis
of variance (ANOVA) using Newman–Keuls test for post hoc compari-
sons and Bonferroni correction. The relationship of spatial memory,
with muscarinic receptor binding, CHRM2 gene expression and
ChAT in different brain regions at different time points was evaluated
by Pearson correlation test. The two tailed p value below 0.05 was
considered significant. The statistical tests were performed using SPSS
v.16 (SPSS Inc., Chicago, IL).
Radioligand (concentration) Competitor (concentration)
3
H-Quinuclidinyl benzylate (1 × 10−9 M) Atropine sulfate (1 × 10−6 M)
 P.S. Chauhan et al. / Physiology & Behavior 156 (2016) 148–155
4. Results
4.1. Clinical observation
From 6 dpi, the rats started showing symptoms which included
sluggish-ness, somnolence, pelvic elevation while walking, lethargy,
huddling in corner and slight hind limb disability which persistent till
33 dpi. The control rats remained asymptomatic.
4.2. Spatial learning and memory
Spatial learning and memory showed decline in rats inoculated with
JEV on 10 and 33 dpi. A significant decrease in the spatial memory
(47.5%, p b 0.01; 30.2%, p b 0.01) was observed on 10 and 33 dpi com-
pared to control rats (Fig. 2). However a trend of recovery in the spatial
memory was observed on 48 dpi.
4.3. Muscarinic–cholinergic receptors in brain regions
Significant decrease were found in the binding 3H-QNB to frontal cor-
tex (31%, p = 0.001; 26%, p = 0.003), hippocampus (57%, p = 0001;
39.9%, p = 0.002), cerebellum (31.2%, p = 0.008; 21.6%, p = 0.007) on
10 dpi and 33 dpi as compared to control rats. However, in corpus stria-
tum there was insignificant decrease (9.30%, p = 0.456; 8.9%, p = 0.30)
on 10 dpi and 33 dpi. The decrease activity of 3H-QNB binding were more
marked on day 10 compared to day 33 in frontal cortex, hippocampus
and cerebellum. On 48 dpi, 3H-QNB binding in frontal cortex, hippocam-
pus, cerebellum and corpus striatum (0.46%, 1.4%, 13.2%, 4%) remained
low compared to controls (Table 2). The decrease in the binding of cho-
linergic–muscarinic receptors in frontal cortex, hippocampus cerebellum
and striatum was observed in the cortical membrane of these brain re-
gions was due to alterations in the number of binding sites (Bmax) as
assessed by the Scatchard analysis (data not shown). A trend of recovery
was observed in 3H-QNB binding in frontal cortex, hippocampus and cer-
ebellum in JEV inoculated rats compared to controls (Fig. 3).
4.4. mRNA expression CHRM2 receptor gene
A significant decrease in the expression of CHRM2 in frontal cortex
(48%, p b 0.001; 38%, p b 0.01), hippocampus (43%, p b 0.001; 37%,
p b 0.05) and cerebellum (46%, p b 0.001; 42%, p b 0.05) was observed
in JEV inoculated rats compared to controls (Fig. 4). These significant
changes were not observed on 48 dpi, except in cerebellum (34%,
p = 0.05). However, in striatum no significant changes were observed
at any time point.
Fig. 2. Spatial memory and learning by Y maze on 10, 33 and 48 dpi in rats inoculated with
106 pfu/ml JEV in 12 day old Wistar rats. Values are mean ± SEM of five animals in each
group. dpi = days post-inoculation, JEV = Japanese encephalitis virus.
151
4.5. Choline acetyl transferase protein in brain regions
A significant fold decrease in the expression of ChAT protein in fron-
tal cortex (2.11, p b 0.01; 1.12, p b 0.05) and hippocampus (2.2, p b 0.01;
1.41, p b 0.05) was observed in JEV inoculated rats on 10 and 33 dpi. No
significant change on the expression of ChAT protein in frontocortical
and hippocampal region was observed on 48 dpi compared to control
rats. (See Fig. 5.)
4.6. Correlation between ChAT, CHRM2 and total muscarinic receptor
activity with spatial memory
Spatial memory on 10 dpi correlated with ChAT activity on 48 dpi in
hippocampus (r = 0.929, p = 0.02) and CHRM2 on 10 dpi in corpus stri-
atum (0.901, p = 0.03). However on 33 dpi spatial memory was
inversely correlated with total muscarinic receptor binding on 10 dpi
in frontal cortex (r = − 0.999, p = 0.00), 48 dpi in hippocampus
(r =−0.964, p = 0.008) and cerebellum (r = −0.964, p = 0.008), cor-
pus striatum (r = −0.999, p = 0.001) and positively correlation were
found in corpus striatum on 10 and 33 dpi (r = 0.976, p = 0.024 and
r = 0.971, p = 0.029). Moreover spatial memory on 48 dpi were
found to be correlated with ChAT activity in frontal cortex on 10 dpi
(r = 0.924, p = 0.025) and total muscarinic receptor binding in corpus
striatum on 10, 33 and 48 dpi (r = 0.962, p = 0.038, r = 0.994, p =
0.006 and r = − 0.994, p = 0.006). CHRM2 gene in corpus striatum
on 10 dpi were also found to be correlated with spatial memory on
48 dpi (r = 0.925, p = 0.024).
5. Discussion
JEV infected rats revealed impairment in spatial learning and memory
from 10 dpi to 33 dpi. The impairment in memory and learning was asso-
ciated with reduction in expression of ChAT, 3H-QNB and CHRM2 gene.
These changes were followed by recovery after 33 dpi. ChAT is the most
sensitive marker of cholinergic neurons in the CNS. Neurobehavioral
and learning deficits are involved in a number of CNS infections such as
Borna disease virus (BDV) [12,13], rabies [49], vesicular stomatitis virus
[34], West Nile encephalitis virus [45] and herpes simplex encephalitis
virus [3]. In BDV, the behavior and learning deficits occur due to persis-
tently infected cells without accompanying inflammation [9,35].
The neurobehavioral alterations are related to anatomical substrate
and neurotransmitter alterations. Earlier studies have revealed maxi-
mum number of JEV RNA copies were present in midbrain followed by
thalamus, cortex and striatum on 10 dpi and were not detected on
20 dpi. The RNA copies were related to motor deficit [48]. These results
were supported by immunohistochemical studies which also showed
the JEV antigen localization in these brain regions [8,23].
Several studies on JE patients have revealed the cognitive and behav-
ioral impairment. In a study on 55 JE patients followed up for 1–1.5 years
showed mental impairment in 21.8% [24]. In another study on 80 pa-
tients with a follow up of 5 years revealed a wide variety of intellectual
abnormalities [14]. Tropism of JEV to different brain regions is attributed
to specific receptors on the neurons that have strong affinity to JE virus
[17]. Neurotransmitter defect in neurons caused by virus may affect cog-
nitive and learning. The cholinergic system, including the muscarinic
acetylcholine receptors and choline acetyl transferase, play an important
role in memory learning and in early development [18]. These markers
may paly role in JE infection as in Parkinson's disease, AD and in other
viral diseases like HIV, Borna virus and west Nile encephalitis.
In our study the involvement of cholinergic system was evidenced by
significant reduction of ChAT in frontal cortex and hippocampus. Our re-
sults are in agreement with a study in which Lewis rats infected with
BDV showed decrease in the ChAT activity in frontal cortex and hippo-
campus on day 10 (pre-encephalitis) (9% and 11.16%) and between 14
and 22 days (encephalitis) (25.19% and 38.9%) significant reduction as
compared to controls [13]. 3H-QNB and M2 receptors were significantly
152 P.S. Chauhan et al. / Physiology & Behavior 156 (2016) 148–155

Table 2
Values are mean ± SEM of five animals in each group. Values expressed in pmol 3H-QNB bound/g protein in frontal cortex, corpus striatum, hippocampus and cerebellum.

dpi (days Brain regions

post-Inoculation)
Frontal cortex p value Corpus striatum p value Hippocampus p value Cerebellum p value
Mean ± SD (n = 5) Mean ± SD (n = 5) Mean ± SD (n = 5) Mean ± SD (n = 5)
pmol 3 H-QNB bound/g pmol 3 H-QNB bound/g pmol 3 H-QNB bound/g pmol 3 H-QNB bound/g
protein protein protein protein

10 dpi control 430.60 ± 51.2 0.001 288.40 ± 67.19 0.461 452.60 ± 52.09 0.001 191.80 ± 34.66 0.008
10 dpi JEV 298.40 ± 24.78 261.20 ± 38.76 215.00 ± 84.84 131.80 ± 15.35
33 dpi control 458.20 ± 39.48 0.003 234.25 ± 26.23 0.296 433.80 ± 49.33 0.002 226.20 ± 20.83 0.007
33 dpi JEV 337.00 ± 52.32 213.80 ± 27.67 260.75 ± 59.80 177.20 ± 22.53
48 dpi control 425.80 ± 30.15 0.903 246.40 ± 27.82 0.625 219.60 ± 23.44 0.825 428.80 ± 48.39 0.032
48 dpi JEV 423.75 ± 12.33 234.80 ± 42.32 190.40 ± 9.099 422.60 ± 36.29

reduced in cortex, hippocampus, striatum and cerebellum. Rats infected
with rabies virus also showed reduced total muscarinic receptors in brain
homogenates. Initially there was increase in 3H-QNB binding on the first
day, which was followed by decrease in 3H-QNB bindings in brainstem,
caudate nucleus, cerebral cortex hippocampus, cerebellum and medulla
as the symptoms of rabies appeared. These results may suggest that
there is alteration of specific neuronal functions although cell viability
is not lost [49]. In our study 3H-QNB binding was reduced, suggesting a
different mechanisms of injury in rabies and JE. We did not note any
rise in 3H-QNB binding in our study because our observation was not
done on the first day after inoculation of JE virus. Amongst the CNS
viral infections HIV/AIDS is associated with dementia. An experimental
study using Simian immunodeficiency virus (SIV) infected monkeys re-
vealed reduction of ChAT in putamen and hippocampus during symp-
tomatic infection. In animal studies with AIDS, the ChAT activity was
further reduced. The reduction of ChAT was not related to viral load or
CNS pathological lesions suggesting that cognitive dysfunction may
occur early in immune deficiency viral infection [10].
On 33 dpi, spatial memory and learning were significantly impaired
compared to control. ChAT and CHRM2 expression was also decreased
on 33 dpi. However, on 48 dpi spatial memory and learning did not
show any significant change as the expression of ChAT, CHRM2 and
muscarinic receptors also did not show any significant decrease
compared to controls. This may be attributed to spontaneous recovery
from JEV infection.
The correlation between ChAT, 3H-QNB binding and M2 receptors
suggest the important role of cholinergic functions in memory and
learning in JEV infection. Cholinergic neurons are present in basal
forebrain bundle, which project to frontal parietal and temporal cortex
and are responsible for memory and learning. Cholinergic neurons in

Fig. 3. Expression of cholinergic muscarinic receptor in frontal cortex, striatum, hippocampus and cerebellum on 10, 33 and 48 dpi. Values are mean ± SEM of five animals in each group.
dpi = days post-inoculation, JEV = Japanese encephalitis virus.
 P.S. Chauhan et al. / Physiology & Behavior 156 (2016) 148–155 153

Fig. 4. CHRM2 mRNA gene expression in frontal cortex, striatum, hippocampus and cerebellum of rats on 10, 33 and 48 dpi. Values are mean ± SEM of five animals in each group. dpi =
days post-inoculation, JEV = Japanese encephalitis virus. *Significantly differs from control (p b 0.05).

neostriatum (caudate and putamen) are focal circuit neurons and have
a role in motor regulation. Pontomesencephalic neurons project to
many areas such as thalamus, neostriatum, basal forebrain bundle and
other mesopontine areas and have a role in arousal and cardiovascular
respiratory functions. In JE patients, a variety of cognitive, behavioral
and movement disorders are reported [14,21]. The involvement of thal-
amus, striatum, brainstem and cerebral cortex are typically involved in
JE as revealed in autopsy and MRI studies [20,25,32,44]. Thalamus,
brainstem, corpus striatum and cerebral cortex which are involved in
JE also have prominent cholinergic innervation and may contribute to
the observed cognitive and learning deficits.
In neurodegenerative disorders such as AD, the role of cholinergic
neurotransmission has been evaluated in greater details than in
neuro-infection. In AD accumulation of β amyloid (Aβ) is followed by
impaired muscarinic regulation which leads to impairment of memory
and learning [38,51]. In AD there is selective degeneration of cholinergic
neurons in basal forebrain corresponding to significant loss of nicotinic
receptors and certain types of muscarinic receptors in cortical and
hippocampal regions of brain [5,7,11,15,40].
In the brain regions affected by JE, there may be reduction of cholin-
ergic activity with reduction of receptors as was observed in our study.
All the markers of cholinergic transmission, ChAT, total muscarinic
receptors and M2 receptors were significantly reduced which was
consistent with damage of neurons. In an earlier study we have reported
reduction of catecholamine in different brain regions [33] and D2 recep-
tors in experimental model of JE (unpublished observations). In the
present study, after 33 dpi there was increase in ChAT, total muscarinic
receptor and M2 receptor expression along with improvement in
154 P.S. Chauhan et al. / Physiology & Behavior 156 (2016) 148–155

Fig. 5. Expression of ChAT protein by Western blot on 10, 33 and 48 dpi in rat hippocampus and frontal cortex after intracerebral inoculation of JEV 106 pfu/ml in 12 day old Wistar
rats. dpi = days post-inoculation, JEV = Japanese encephalitis virus.

memory and learning. Such recovery is supported by clinical studies already been reported. This study is based on intracerebral inoculation
in JE patients. In JE after initial worsening of consciousness and neuro- and other routes of JEV inoculation have not been studied. These limita-
logical worsening, the patients recover with respect to neurological tions can be addressed in a future study.
dysfunction, behavior and cognition [14].
Our study for the first time provides evidence of role of cholinergic
transmission in the memory and learning impairment in a rat model Conflicts of interest
of JE. In this study we have not evaluated the relationship between
viral dose (RNA copies or viral protein) but these experiments have There are no conflicts of interest to declare.
 P.S. Chauhan et al. / Physiology & Behavior 156 (2016) 148–155
Funding support
Yes, this study was (No. CST/SERPD/D-996) financially supported by
a grant from Uttar Pradesh Council of Science & Technology Lucknow,
UP, India.
Acknowledgment
We thank Mr. Rakesh Kumar Nigam for secretarial help.
Ethical approval no
KGMU/49/IAEC/2013
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