Neurochem Res (2013) 38:2503–2515
DOI 10.1007/s11064-013-1163-4
ORIGINAL PAPER
Psychoneurochemical Investigations to Reveal Neurobiology
of Memory Deficit in Epilepsy
Awanish Mishra • Rajesh Kumar Goel
Received: 25 July 2013 / Revised: 19 September 2013 / Accepted: 26 September 2013 / Published online: 8 October 2013
Ó Springer Science+Business Media New York 2013
Abstract Pentylenetetrazole-kindling induced memory
deficit has been validated in our previous study. The
present study attempts a neurochemical investigation to
reveal possible targets for treatment of memory deficit
associated with pentylenetetrazole-kindling. Kindling was
induced by administering subconvulsive dose of penty-
lenetetrazole (35 mg/kg; i.p.) at an interval of 48 ± 2 h.
Successfully kindled animals were divided into two groups
(interictal and postictal group), while non-kindled (naive)
animals served as naı̈ve group. In postictal group, animals
were challenged with pentylenetetrazole (35 mg/kg) on
days 5, 10, 15 and 20. Learning and memory were evalu-
ated in all experimental groups using elevated plus maze
and passive shock avoidance paradigm on days 5, 10, 15
and 20. After behavioral evaluations on day 20, all animals
were sacrificed to remove their brains. Neurochemical
(glutamate, GABA, norepinephrine, dopamine and seroto-
nin) changes and acetylcholinesterase activity and total
nitrite level were estimated using HPLC-FD methods and
microplate reader method respectively, in discrete brain
parts. The results of the neurochemical estimation dem-
onstrated the imbalance in excitatory/inhibitory tone,
reduction in monoamine level, elevated nitrosative and
acetylcholinesterase activity in the cortex and hippocam-
pus, as responsible factors for the pathobiology of learning
and memory deficit in epilepsy. Restoration of these
changes may be targeted for the management of memory
deficit in epileptic patients.
A. Mishra
R. K. Goel (&)
Department of Pharmaceutical Sciences and Drug Research,
Punjabi University, Patiala 147002, Punjab, India
e-mail: goelrkpup@gmail.com
Keywords Pentylenetetrazole-kindling
Epileptic
comorbidity
Learning and memory deficit

Neurotransmitters
Post and interictal status
Introduction
Epilepsy is a heterogeneous biomedical disorder, with
enormous variations in etiology and clinical features,
resulting in irregular episodic bursts of electrical activity in
certain neurons, which may spread to the entire brain. It is
one of the most common serious neurological disorders in
the world [1]. Chronic epileptic patients are more often
associated with one or more neurobehavioral comorbidi-
ties. Out of which, learning and memory deficit emerges as
one of most debilitating neurobehavioral comorbidity of
chronic epilepsy [2, 3] affecting around 30 % of patients
with epilepsy [4] and greatly contribute to the major
adverse effect on overall health and quality of life and
substantially increase health care costs [5].
Cognitive functions in epileptic patients are as diverse as
the epileptic condition itself by virtue of their origin,
topography of epileptogenic foci, pathological mechanisms
and various features characterizing the clinical epilepsy, all
contribute to the deleterious effects on cognition. Impaired
cognitive outcome is generally associated with an early
onset and a long duration of the disease and with poor
seizure control [6].
Management of epilepsy is ineffective with available
antiepileptic drugs, showing about 70 % effectiveness in
patients with epilepsy [6], which poses a significant risk
factor for progressive memory deficit in these patients.
Further adding to this woe, most of the available antiepi-
leptic drugs, used to manage epilepsy may also cause
memory deficit [7–10]. Owing to the risks of seizurogenic
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potential concerned with management of cognitive
comorbidities with conventional memory enhancing drugs
[11], these patients often remain untreated. Therefore,
clinical interventions to prevent these commodities in
patients with epilepsy are still issues of substantial concern.
Disturbances in coordination of neurotransmission in
different brain regions during epilepsy may lead to memory
deficit. Thus it is hypothesized that examining the effect of
neurochemical alterations on learning and memory deficit
following pentylenetetrazole-kindling may help to under-
stand the neurobiology of susceptibility to learning and
memory deficit in epileptic patients and progressive
learning and memory deficit in uncontrolled or untreated
patients with epilepsy, to provide novel therapeutic targets
for the management of memory deficit in epileptic patients.
In chronic epileptic patients, ictal and postictal states are
relative momentary than interictal state, which persists for
longer time. As the time span of the interictal state prevails
over postictal state, the monitoring of behavioral and
neurochemical changes in the interictal state may be cru-
cial to provide valuable insight for the pathology of sus-
ceptibility of learning and memory deficit in epilepsy.
While for understanding the neurobiology of progressive
learning and memory deficit in uncontrolled seizures neu-
rochemical changes may be monitored after multiple con-
vulsive episodes.
Chemical or electrical kindling in rodents are regarded
as a model of human temporal lobe epilepsy. Kindling is a
phenomenon in which repeated and intermittent subcon-
vulsive stimulus resulting in progressive stimulus-induced
convulsions culminating to generalized seizures. Penty-
lenetetrazole noncompetitively blocks GABA-mediated
Cl- influx through allosteric modulation at Cl- channel,
leading to neuronal depolarization and propagation of sei-
zures. Pentylenetetrazole-kindling model also appears dis-
tinctive in providing opportunity to study progressive
cognitive changes with a close resemblance to clinical
epilepsy [12]. Therefore the present study was executed to
explore the relationship between memory deficit and neu-
rochemical alterations in discrete brain areas to delineate
the possible targets for comprehensive management of this
problem in future using pentylenetetrazole-kindling model.
Methods
Drugs and Chemicals
Pentylenetetrazole, DTNB, Griess reagent, 5-Hydroxytrip-
tamine was procured from Sigma-Aldrich, Co., St. Louis,
MO, USA, Glutamate and GABA from Loba Chemie,
Mumbai. Norepinephrine (a gift sample from Troikaa
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Neurochem Res (2013) 38:2503–2515
Pharmaceuticals, Dehradun, Uttarakhand, India), Dopa-
mine and o-phthaldehyde (HiMedia, Mumbai) and Phe-
nytoin Sodium from Jackson Laboratories Ltd., India All
other reagents were of the highest purity available and
HPLC grade water was procured from our laboratory (Lab
Pure, Bio Age, India) and filtered through Millipore
Membrane (0.45 lm) filtered (Millipore, USA).
Animals
The studies were carried out on male Swiss Albino mice
(22–28 g weight) and were obtained from the breeder
(Chaudhary Charan Singh Haryana Agricultural Univer-
sity, Hisar, Haryana, India). Swiss Albino mice were
housed in groups of 10 mice/cage in standard cages in a
room temperature of 22 ± 2 °C, under natural light/dark
cycle and had free access to water and food (standard
laboratory pellets) before the experiments. The mice were
acclimatized at lab conditions for 5 days before the start of
the experiment. Each experimental group consisted of 10
animals. All the experimental work had been carried out
from 08:00 to 16:00 h. The experimental protocol was duly
approved by the Institutional Animal Ethics Committee
(IAEC) and the care of the animals was carried out as per
the guidelines of the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA),
Ministry of Environment and Forests, Government of India
vide protocol approval no. 107/99/CPCSEA/-2009-4.2.
Induction of Kindling
Induction of kindling was done by the method previously
validated in our laboratory [12]. Briefly, pentylenetetraz-
ole (dissolved in warm saline) was injected intraperito-
neally in a volume of 10 ml/kg mice at a sub-convulsive
dose of 35 mg/kg at 48 ± 2 h intervals. After each
injection, the mice were placed individually into Plexiglas
cages (20 9 20 9 30 cm) and observed for 30 min. The
intensity of the convulsions was registered according to
modified Racine’s scale [12]; stage 0: no response; stage
1: hyperactivity, restlessness and vibrissae twitching;
stage 2: head nodding, head clonus and myoclonic jerks;
stage 3: unilateral or bilateral limb clonus; stage 4: fore-
limb clonic seizures; stage 5: generalized tonic–clonic
seizures with falling; stage 6: hind limb extensor and
death was considered as stage 7. The transition of the
convulsion intensity from the 4th to 5th degree reflected
the generalization of the convulsive activity, manifested
by the tonic–clonic convulsions. The animals were con-
sidered kindled on appearance of stage 5 (tonic–clonic
convulsion) after two consecutive pentylenetetrazole
administrations.
Neurochem Res (2013) 38:2503–2515
Fig. 1 Schematic presentation
of experimental protocol
Experimental Protocol
A total of 40 animals were employed in this study. The
group I: naı̈ve animals, consisted of naı̈ve animals (non
kindled, n = 10) and rest 30 animals were subjected to
pentylenetetrazole-kindling. The animals showing resis-
tance to pentylenetetrazole-kindling were excluded from
study and only successfully kindled animals were further
incorporated in this study and randomly divided into two
groups. Group II: postictal group animals: consisted of
kindled animals (n = 10) subjected to frequent convulsive
episodes by administering subconvulsive dose of pentylen-
etetrazole (35 mg/kg; i.p.) on day 5, 10, 15 and 20. Group
III: interictal group animals: consisted of kindled animals
(n = 10) without frequent convulsive episodes (Fig. 1).
In postictal group frequent convulsions were induced
(with pentylenetetrazole challenging dose) in order to mimic
pathology of uncontrolled seizures (postictal state) in epi-
leptic patient where intermittent seizures occurs. Whereas
interictal group providing insight of interictal state pathol-
ogy of epileptic patients where next episode of seizure is
unpredictable. Behavioral evaluations were done in all the
groups on day 5, 10, 15 and 20 (in postictal group behavioral
evaluations were done after 2 h of pentylenetetrazole chal-
lenging dose, when their locomotor activity becomes nor-
malized). On day 20, after behavioral evaluations, animals
were decapitated for neurochemical analysis.
Behavioral Assessments
Transfer latency in Elevated Plus Maze
Spatial memory was evaluated using elevated plus maze on
days 0, 10, 15 and 20 following the procedure previously
standardized in our laboratory [12]. The elevated plus maze
for mice consisted of two open arms (16 cm 9 5 cm) and
two closed arms (16 cm 9 5 cm 9 12 cm) and maze was
elevated to a height of 25 cm from floor. Each mouse was
gently placed at the distal end of an open arm of the
apparatus facing away from the central platform and the
2505
time it took for the mice to move from the open arm to
either of the enclosed arms (transfer latency) was recorded.
The criterion of an animal’s entry into the enclosed arm
was crossing with all four legs of an imaginary line sepa-
rating the enclosed arm from the central space. After
entering the enclosed arm, the mice were allowed to move
freely in the maze regardless of open and enclosed arms for
20 s. Then, the rat was returned to its home cage. Retention
was examined on day 5, 10, 15 and 20. To remove any
confounding olfactory cues the maze was cleaned with
alcohol–water solution after each mice.
Number of Mistakes and Step Down Latency in Passive
Shock Avoidance Paradigm
For the evaluation of contextual fear memory, modified
passive shock avoidance paradigm previously standardized
in our laboratory [12] was used. The apparatus consisted of
a Plexiglas box (27 cm 9 27 cm 9 27 cm) with a grid
floor (3 mm stainless steel rods set 8 mm apart), having a
shock free zone (non-conducting platform 10 cm 9
7 cm 9 1.7 cm) in the center of the grid floor. Electric
shock (20 V AC) was delivered to the grid floor. Each
mouse was trained to stay on the shock free zone for at
least 120 s, for this the animals were gently placed on the
shock free zone, when the mouse stepped down placing all
its paws on the grid floor, shocks were delivered for 15 s.
The process was repeated till the animal learned to stay on
shock free zone for at least 120 s and number of trials was
counted on day 0. The retrieval of learned task was eval-
uated by recording the changes in the number of mistakes
and step down latency on day 5, 10, 15 and 20.
Neurochemical Estimations
After behavioral evaluation on day 20 (4 h after last
pentylenetetrazole injection in postictal group) all animals
were sacrificed by cervical dislocation and their brains
were dissected to isolate different brain regions (cortex,
hippocampus, cerebellum and brain stem). Isolated brain
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parts were weighed and subdivided into two equal portions.
One portion was homogenized in ice cold 10 % w/v
(0.1 M) perchloric acid and centrifuged at 14,000 g for
30 min at 4 °C (REMI C-24BL, Cooling Centrifuge,
REMI, India) and clear supernatants were used for esti-
mation of amino acids (glutamate and GABA), monoam-
ines (noradrenaline, dopamine and serotonin). While
second portion was homogenized in ice cold 10 % w/v
(0.05 M, pH 7.4) phosphate buffer and centrifuged at
6,000 g for 20 min at 4 °C and clear supernatant was used
for estimation of total nitrite level and acetylcholinesterase
activity.
Estimation of Glutamate and GABA by HPLC-FD Method
In this study the rapid estimation of glutamate and GABA
was done using HPLC-FD method previously standardized
in our laboratory [13]. The method is based on the con-
ventional derivatization technique which involves most
commonly used derivatizing agent o-phthalaldehyde.
o-phthalaldehyde reacts with primary amines in the pre-
sence of thiol and generates derivatives which are both
electro active and fluorescent.
The instrumentation of Waters HPLC system (Milford,
USA) for analysis of glutamate, GABA and monoamines
were similar and consisterd of 515 binary pumps (Waters,
USA), 2475 Fluorescent detector (Waters, USA) and
Rheodyne manual injector (20 lL). The chromatographic
separation was achieved on Zorbex SB-C18, reversed-
phase column (4.6 mm 9 150 mm, 5 lm) (Agilent, USA).
Chromatographic analyses were performed at room tem-
perature. The data were acquired and processed in the
Empower ProÒ Operating System (WatersÒ, Milford,
USA).
The mobile phase consisted of 100 mM disodium
hydrogen phosphate and methanol in 60: 40 ratios. The pH
of the mobile phase was maintained to 6.70 ± 0.01 using
o-phosphoric acid and then filtered through a 0.45 lm
membrane (Millipore, USA) and degassed (Transsonic T
570/H, Elma, Germany). The flow rate was set to 1 mL/
min. The stock derivatizing solution was prepared by dis-
solving 27 mg of o-phthaldehyde in 1 ml of HPLC grade
methanol. b-mercapto ethanol (5 lL) was added to this
solution and volume made up to 10 ml using 0.1 M sodium
tetraborate (pH 9.3). For the preparation of working de-
rivatizing solution, 2.5 mL of the stock derivatizing solu-
tion was mixed with 7.5 ml of 0.1 M sodium tetraborate
buffer. This solution was prepared and utilized freshly. The
derivatisation was performed by mixing 100 lL sample or
standard solutions with 100 lL of working derivatizing
solution and was vortex for a minute. The resulting solution
was filtered through a sample filter (0.45 lm, Millipore,
USA) and injected to HPLC.
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One milligram per milliliter stock solutions of GABA/
glutamate standards were prepared in HPLC grade water,
aliquoted out and stored at -20 °C. The standard curve of
glutamate and GABA was plotted using these concentrations
and corresponding area under the curve. Glutamate/GABA
was detected with at the excitation wavelength 345 nm and
an emission wavelength of 442 nm and their peaks were
identified by comparing their retention time in the standard
and sample and amount of glutamate/GABA was quantified
using area under the peak of corresponding sample using
their straight line equation. The straight line equation
was derived (Glutamate: y = 44187x ? 1E ? 06, R2 =
0.9987 and GABA: y = 219217x - 6E ? 06; R2 =
0.9964; where y: area under peak and x: concentration in ng/
mL) and utilized further to determine the different concen-
tration of glutamate and GABA in the samples. The results
were expressed as ng/g of wet tissue.
Estimation of Noradrenaline, Dopamine and Serotonin
by HPLC-FD Method
Estimation of the monoamines in the brain samples was
done using HPLC-FD method previously standardized in
our laboratory with slight modification [13]. HPLC
instrumentation and column was similar as that of gluta-
mate/GABA estimation while slight modification has been
mentioned below.
The mobile phase consisted of sodium acetate (0.02 M),
methanol (16 %), heptane sulfonic acid (0.137 %), EDTA
(0.2 mM), and dibutylamine (0.1 %, v/v). The solution was
adjusted to pH 3.92 ± 0.01 with o-phosphoric acid and
filtered through a 0.45 lm membrane (Millipore, USA) and
degassed (Transsonic T 570/H, Elma, Germany). The flow
rate was set to 1 ml/min.
Norepinephrine, dopamine and serotonin were detected at
the excitation wavelength 280 nm and emission wavelength
of 315 nm. Monoamine peaks were identified by comparing
their retention time in the standard and sample and the
concentration of the norepinephrine, dopamine and seroto-
nin was estimated according to their area under a curve using
their straight line equation. The straight line equation was
derived (norepinephrine: y = 124373x ? 255920, R2 =
0.9981; dopamine: y = 70355x ? 113,535; R2 = 0.9938;
serotonin: y = 151725x - 446,462; R2 = 0.997; where x:
concentration in ng/ml and y: area under peak) and utilized
further to determine the different concentration of norepi-
nephrine, dopamine and serotonin in the samples. The results
were expressed as ng/g of wet tissue.
Estimation of Nitrite Level Using Microplate Method
Nitrite is the stable end product of nitric oxide in vitro
systems. Accumulation of total nitrite was measured in
Neurochem Res (2013) 38:2503–2515
cell-free supernatants from brain homogenate using
microplate reader method, using Griess reagent, previously
standardized in our laboratory [14]. The concentration of
total nitrite was estimated using the straight line equation
of nitrite (y = 0.0008 x ? 0.0046; R2 = 0.9961; where x:
concentration in ng/ml and y: area under peak). The results
were expressed as ng/g of wet tissue.
Estimation of Brain Acetylcholinesterase Activity
For the estimation of brain acetylcholinesterase (AChE)
activity, the method previously described by Ellman and
his colleagues [15] with slight modification. Briefly, 40 ll
of filtered brain homogenate was mixed with 80 ll of
Ellman Reagent (39.6 mg DTNB and 15 mg NaHCO3
dissolved in 10 ml 50 mM phosphate buffer pH 7.4) and
200 ll of 50 mM phosphate buffer (pH 8) in 96 well plate
and shaken properly. The absorbance of the reaction mix-
ture was recorded prior to addition of substrate at 412 nm.
The reaction was initiated by adding 10 ll of the enzyme
substrate (10 mM Acetylthiocholine iodide) to each well
and was allowed to incubate for 15 min. Yellow color
developed and the solution was read at 412 nm using
Microplate Reader (APR-4 Microplate Reader, Logotech,
ISE Group, Germany). A molar extinction coefficient of
14,150 M-1 Cm-1 was used to calculate enzyme activity.
Enzyme activity was expressed as lM of acetylcholine
hydrolyzed per milligram of wet tissue.
Statistical Analysis
The statistical analysis was performed using the Sigma Stat
Statistical Software version 3.5. In the behavioral estima-
tions, comprising of two variables (different groups and
different days), the intergroup and intra group variation
was measured by two-way analysis of variance (ANOVA)
followed by Student–Newman–Keuls Test (for multivariate
analysis). While for the biochemical estimation (compris-
ing of one variable i.e. different treatment) one-way
ANOVA followed by Student–Newman–Keuls Test was
applied. In the result each value was expressed as
mean ± SEM of ten animals and statistical significance
was considered at P \ 0.05.
Results
Kindling was successfully induced in the mice by the
repeated administration of subconvulsive dose of penty-
lenetetrazole in mice. Average number of pentylenetet-
razole injections required to induce successful kindling
state in mice was found to be 17 ± 3. Animals showing
resistance to pentylenetetrazole-kindling were excluded
2507
from study and successfully kindled animals were included
in the study. Tonic–clonic seizures (stage 5 of modified
Racine’s Scale) were observed in animals of postictal
group after administration of pentylenetetrazole challeng-
ing dose on day 5, 10, 15 and 20.
Effect on Transfer Latency in Elevated Plus Maze
The significant change in transfer latency was observed on
day 0 (F(2,27) = 49.799; P \ 0.001), day 5 (F(2,27) =
66.926; P \ 0.001), day 10 (F(2,27) = 161.337; P \ 0.001),
day 15 (F(2,27) = 167.550; P \ 0.001) and day 20
(F(2,27) = 151.450; P \ 0.001). Naı̈ve animals had shown a
tendency to decrease transfer latency on days 5, 10, 15 and
20. There was a significantly increased in transfer latency of
pentylenetetrazole-kindled mice (of both postictal and
interictal group) as compared to naı̈ve mice on days 0. On
day 5, 10, 15 and 20 the transfer latency of post and interictal
group mice was significantly higher than that of their
transfer latency on day 0 (P \ 0.05) and naı̈ve group on
respective days (P \ 0.001). However transfer latency of
the postictal and interictal group was not significantly dif-
ferent than each other on different days (Fig. 2).
Effect on Number of Mistakes and Step Down Latency
in Passive Shock Avoidance Paradigm
There was significant change in number of mistakes
observed in different groups on day 5 (F(2,27) = 49.457;
P \ 0.001), day 10 (F(2,27) = 42.106; P \ 0.001), day 15
(F(2,27) = 45.275; P \ 0.001) and day 20 (F(2,27) = 84.986;
P \ 0.001). Naı̈ve group animals have shown requirement
of average 2.5 ± 0.22 numbers of trials to learn to stay for at
least 120 s on shock free zone in passive shock avoidance
paradigm. While pentylenetetrazole-kindled animals (of
both postictal and interictal group) required significantly
Fig. 2 Effect on transfer latency in elevated plus maze. Each value is
expressed as Mean ± SEM. *As compared to naı̈ve, # as compared to
postictal group, ! as compared to day 0 or day 5 data. The significance
level was considered at P \ 0.05 (Student–Newman–Keuls Test)
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Fig. 3 Effect on number of mistakes in passive shock avoidance
paradigm. Each value is expressed as Mean ± SEM. *As compared
to naı̈ve, # as compared to postictal group. The significance level was
considered at P \ 0.05 (Student–Newman–Keuls Test)
increased number of trials 8.5 ± 0.6 (P \ 0.001) than naı̈ve
animals on day 0. On day 5, 10, 15 and 20 there was a
significant increase in the number of mistakes in postictal
and interictal group than that of the naı̈ve group on the
respective days. There was also significant increase in
number of mistakes in postictal group (P \ 0.001) than that
of interictal group on day 5, 10, 15 and 20 (Fig. 3).
The significant change in step down latency was
observed in different groups on day 5 (F(2,27) = 253.490;
P \ 0.001), day 10 (F(2,27) = 248.199; P \ 0.001), day 15
(F(2,27) = 430.948; P \ 0.001) and day 20 (F(2,27) =
290.727; P \ 0.001). Naı̈ve group animals had shown
average step down latency of 120 s on day 5, 10, 15 and 20.
Post-hoc analyses indicated a significant decrease in step
down latency of the postictal group (P \ 0.001) and
interictal group (P \ 0.001) as compared to the naı̈ve
group on day 5, 10, 15 and 20. On day 15 and 20 the step
down latency of postictal group was significantly reduced
(P \ 0.001) than that of interictal group (Fig. 4).
Neurochemical Changes
While analyzing amino acids using the HPLC-FD method,
glutamate and GABA using HPLC-FD method glutamate
appeared at 2.72 min and GABA at 13.60 min of retention
time. However in case of monoamine estimation nor-
adrenaline eluted first (retention time = 2.89 min) fol-
lowed by dopamine (retention time = 4.68 min) and
serotonin (retention time = 9.16 min).
Changes in Glutamate/GABA Ratio
The Glutamate and GABA ratio was calculated as the indices
of excitatory and inhibitory neurotransmitter ratio in discrete
areas of the brain. There was significant difference between
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Fig. 4 Effect on step down latency in passive shock avoidance
paradigm. Each value is expressed as Mean ± SEM. *As compared
to naı̈ve, # as compared to postictal group, ! as compared to day 5
data. The significance level was considered at P \ 0.05 (Student–
Newman–Keuls Test)
the groups in cortical (F(2,27) = 76.740; P \ 0.001), hippo-
campal (F(2,27) = 66.263; P [ 0.001), cerebellum (F(2,27) =
27.925; P \ 0.001) and brain stem (F(2,27) = 39.836;
P [ 0.001) glutamate/GABA ratio. In postictal group, cor-
tical glutamate/GABA ratio was significantly reduced
(P \ 0.001) however, in interictal group cortical glutamate/
GABA ratio was significantly increased (P \ 0.001) as
compared to naı̈ve animals. There was around two fold
increase in cortical glutamate/GABA ratio was observed in
interictal group animals as compared to postictal group
animals. Hippocampal glutamate/GABA ratio was unchan-
ged in the postictal group while significantly increased
(P \ 0.001) in interictal group as compared to naı̈ve ani-
mals. The hippocampal glutamate/GABA ratio was around
two times increased in interictal group animals as compared
to postictal animals. Significant reduction in cerebellar glu-
tamate/GABA ratio was observed in postictal and interictal
groups as compared to naı̈ve animals. In the brainstem
region, significant reduction (P \ 0.001) in glutamate/
GABA ratio in postictal group while significant elevation
(P \ 0.001) in glutamate/GABA ratio was recorded in
interictal group as compared to naı̈ve animals (Table 1).
Changes in Noradrenaline Levels
There was a significant difference in concentration of nor-
adrenaline in cortex (F(2,27) = 8.951; P \ 0.001), hippocam-
pus (F(2,27) = 106.334; P \ 0.001), cerebellum (F(2,27) =
152.987; P \ 0.001) and brain stem (F(2,27) = 357.208;
P \ 0.001) in between the groups. Post-hoc test (Student–
Newman–Keuls test) indicated a significant decrease in cortical
noradrenaline level of interictal group (P \ 0.01) as compared
to naı̈ve mice, while no significant change in postictal group
was observed. In hippocampus noradrenaline level was
 Table 1 Comparative account of neurochemical changes in different experimental groups
Groups NoradrenalineW DopamineW SerotoninW GlutamateW GABAW Glutamate/ Nitrite levelw AChE activity
Neurochem Res (2013) 38:2503–2515

GABA

Cortex
Naı̈ve 77.25 ± 8.49 551.89 ± 28.63 1,070.07 ± 112.82 6,276.81 ± 234.55 1,743.87 ± 105.08 3.62 ± 0.08 582.08 ± 61.54 153.31 ± 7.26
Postictal 94.62 ± 6.85 129.51 ± 23.96* 255.16 ± 30.86* 4,522.37 ± 375.93* 1,916.69 ± 124.33 2.35 ± 0.04* 814.23 ± 27.51** 279.95 ± 4.11***
Interictal 53.83 ± 4.63*,# 177.38 ± 15.54* 182.63 ± 56.48** 6,045.48 ± 22.42# 1,385.78 ± 46.05*,# 4.36 ± 0.18*,# 770.21 ± 25.95** 185.29 ± 9.28**,####
Hippocampus
Naı̈ve 230.08 ± 4.44 90.27 ± 6.43 826.86 ± 14.52 4,306.83 ± 238.80 1,790.15 ± 42.30 2.43 ± 0.19 419.58 ± 25.95 162.54 ± 7.03
Postictal 172.18 ± 12.13*** 39.66 ± 8.81* 30.87 ± 11.65** 4,744.37 ± 67.15 2,031.12 ± 203.97 2.44 ± 0.21 745.37 ± 47.04*** 210.11 ± 9.56**
Interictal 71.31 ± 3.91**,# 55.73 ± 1.64**,# 154.77 ± 48.93*,# 6,779.23 ± 111.58*,# 1,230.64 ± 41.09*,# 5.51 ± 0.25*,# 812.24 ± 67.82*** 198.25 ± 8.82**
Cerebellum
Naı̈ve 174.34 ± 5.03 403.99 ± 63.11 710.19 ± 72.79 6,877.53 ± 183.53 1,873.18 ± 76.14 3.68 ± 0.16 482.08 ± 37.83 162.11 ± 9.28
Postictal 71.68 ± 2.35** 26.48 ± 2.98** 271.45 ± 11.62* 4,491.10 ± 4.93* 1,959.68 ± 25.13 2.29 ± 0.13* 798.21 ± 5.59*** 246.67 ± 9.55***
Interictal 155.57 ± 5.27*,# 48.49 ± 3.32** 56.02 ± 4.76**,# 5,944.92 ± 41.26*,# 1,822.83 ± 24.46*,# 3.26 ± 0.11*,# 615.74 ± 25.49***,## 210.25 ± 8.22**,#
Brainstem
Naı̈ve 412.82 ± 10.55 1,603.59 ± 20.28 1,241.77 ± 9.95 6,765.27 ± 47.05 2,971.05 ± 58.26 2.28 ± 0.13 477.92 ± 27.51 237.67 ± 8.82
Postictal 109.06 ± 5.58** 602.29 ± 48.09* 1,084.63 ± 7.93* 4,707.58 ± 213.36* 2,705.71 ± 50.13* 1.74 ± 0.19* 485.12 ± 13.98 269.63 ± 6.83*
Interictal 138.14 ± 9.67*,# 89.51 ± 8.51**,# 252.57 ± 49.43**,# 5,796.48 ± 25.13*,# 1,575.96 ± 48.22*,# 3.68 ± 0.15*,# 478.92 ± 12.67 275.24 ± 9.55*
* #
Each value is expressed as Mean ± SEM. The significance level was considered at P \ 0.05 (Student–Newman–Keuls Test); significant as compared to naı̈ve; significant as compared to
postictal group (*/# P \ 0.05; **/## P \ 0.01; ***/### P \ 0.001). W: ng/g of tissue; AChE activity was expressed in micro Moles of acetylcholine hydrolyzed per g of tissue
2509

123
2510
significantly decreased in postictal and interictal group
(P \ 0.001) as compared to naı̈ve group. However, the hip-
pocampal noradrenaline level of postictal group was signifi-
cantly higher (P \ 0.001) than that of interictal group animals.
In cerebellum and brain stem region noradrenaline level was
significantly decreased (P \ 0.001) in postictal and interictal
group animals. In cerebellum and brainstem, noradrenaline
level of interictal group was significantly higher (P \ 0.001)
than that of postictal animals (Table 1).
Changes in Dopamine Levels
There was statistically significant difference in concentra-
tion of dopamine in cortex (F(2,27) = 98.136; P \ 0.001),
hippocampus (F(2,27) = 22.751; P \ 0.001), cerebellum
(F(2,27) = 33.649; P \ 0.001) and brain stem (F(2,27) =
636.184; P \ 0.001) in between the groups. The post hoc
test suggested depletion in cortical, hippocampal, cerebel-
lar and brainstem dopamine level of postictal and interictal
group (P \ 0.001) as compared to naı̈ve group. However,
in interictal group animals, dopamine level was signifi-
cantly higher (P \ 0.001) in cortex and hippocampus,
unchanged in cerebellum and reduced in brain stem
(P \ 0.001) as compared to that of postictal animals in
respective areas (Table 1).
Changes in Serotonin Levels
There was significant difference between the groups in the
concentration of serotonin in cortex (F(2,27) = 43.178;
P \ 0.001), hippocampus (F(2,27) = 200.799; P \ 0.001),
cerebellum (F(2,27) = 61.110; P \ 0.001) and brain stem
(F(2,27) = 13.847; P \ 0.001). The Post-hoc test suggested
significant decrease in cortical, hippocampal, cerebellar
and brainstem serotonin level in postictal and interictal
group (P \ 0.001) as compared to naı̈ve animals. In hip-
pocampus increased level of serotonin was observed in
interictal group animals as compared to postictal animals.
However, reduced brain stem serotonin level was recorded
in interictal animals as compared to postictal animals
(Table 1).
Changes in Total Nitrite Levels
There was statistically significant difference in total nitrite
level in cortex (F(2,27) = 87.398; P \ 0.001), hippocampus
(F(2,27) = 176.832; P \ 0.001), cerebellum (F(2,27) =
357.639; P \ 0.001) in between the groups was observed.
However no change in brainstem nitrite level was observed
in different groups (F(2,23) = 0.0411; P = 0.960). The
Post-hoc test suggested significant enhancement of cortical,
hippocampal and cerebellar nitrite level of postictal and
interictal animals as compared to naı̈ve animals. However,
123
Neurochem Res (2013) 38:2503–2515
significant reduction only in cerebellar nitrite level was
recorded in interictal group as compared to postictal ani-
mals (Table 1).
Changes in Acetylcholinesterase Activity
There was statistically significant difference in acetylcho-
linesterase activity in cortex (F(2,27) = 83.614; P \ 0.001),
hippocampus (F(2,27) = 8.409; P \ 0.001), cerebellum
(F(2,27) = 22.044; P \ 0.001) and brain stem (F(2,27) =
5.716; P \ 0.05) in between the groups was observed. The
Post-hoc test suggested significant enhancement of cortical,
hippocampal, cerebellar and brain stem acetylcholinester-
ase activity of postictal and interictal animals (P \ 0.001)
as compared to naı̈ve animals. However, acetylcholines-
terase activity in cortex and brain stem of interictal group
was significantly reduced than that of postictal animals.
However no significant difference in hippocampal and
brain stem acetylcholinesterase activity was observed
between interictal and postictal animals (Table 1).
Discussion
In the present study, first attempt has been made to cor-
relate postictal and interictal learning and memory deficit
and associated neurochemical changes in discrete brain
parts using pentylenetetrazole-kindling model of epilepsy
in search of putative comprehensive or add on target for the
treatment of learning and memory deficit associated with
epilepsy. Our study has demonstrated susceptibility of
learning and memory deficit in interictal group and pro-
gressive learning and memory deficit in postictal group
along with peculiar neurochemical alterations in discrete
brain parts.
Transfer latency in elevated plus maze model corre-
sponds to short term and long term spatial memory; num-
ber of trials required during the trial session of passive
shock avoidance paradigm suggest the learning behavior;
while step down latency indicated contextual fear memory
in this study. Interestingly, we found both types of memory
impairment in postictal and interictal group. Contextual
fear memory was more worsened in postictal group than
interictal group and progressive uncontrolled seizures in
postictal group further worsened contextual fear memory.
These findings are consistent with literature reports sug-
gesting the memory impairment in pentylenetetrazole-
kindled animals [12–14, 16, 17] and progressive memory
deficit with uncontrolled seizures of epileptic patients [18,
19]. The memory deficit in postictal and interictal group on
day 5 was not significantly different than memory deficit
on day 20. It may be assumed that molecular changes on 5
could be similar to that of day 20 therefore similar degree
Neurochem Res (2013) 38:2503–2515
of memory deficit was observed in post/interictal group
animals on day 5 and day 20.
Concurrent alterations in brain circuitry and chemistry
are believed to play a crucial role in epileptogenesis and
learning and memory formation. For better understanding
of the relationship between the pathophysiology of epi-
lepsy induced learning and memory deficit, the neuro-
chemical interplay in different brain parts was monitored.
In our study the pentylenetetrazole-kindling resulted in
significant changes in concentrations of different neuro-
transmitters in discrete brain regions which vary with time
function. While discussing, major emphasis have been
given to the neurochemical changes in brain regions pri-
marily involved in epileptogenesis, learning behavior and
consolidation of memory viz, cortex and hippocampus.
The imbalance between excitatory and inhibitory tone
produced by decreased GABAergic and/or increased
glutamatergic transmission has been considered as patho-
logical factors for the generation of seizures, both in animal
models and in humans [1, 20]. Similarly in our study, the
major remarkable finding was a shift in the balance between
excitatory and inhibitory amino acid concentrations in the
kindled animals. The elevated cortical and hippocampal
glutamate/GABA ratio in interictal group suggests the
reduced seizure threshold. Although we did not find any
spontaneous recurrent seizures in interictal group, but
reduced seizure threshold in of pentylenetetrazole-kindled
animals can be presumed through the appearance of tonic–
clonic seizures in postictal group animals after administra-
tion of subconvulsive dose of pentylenetetrazole. Reduced
cortical and hippocampal glutamate/GABA ratio level was
decreased and simultaneous enhancement in the GABA
level was recorded in postictal group. The reduction in
glutamate/GABA ratio, a theoretical marker of the neuronal
excitation level, might be due to the activation of compen-
satory mechanisms in postictal group. This observation is
consistent with the hypothesis underlining role of distur-
bances in the balance between excitatory and inhibitory
processes as a major factor leading to epileptogenesis [21].
The evidences suggest that the level of glutamate appears to
be enhanced in ictal state leading to precipitation of seizures
and exhaustion of glutamate may be responsible for its
reduced level in hippocampus [22] and cortex of postictal
group animals.
Glutamate has long been implicated in synaptic plasticity
underpinning learning and memory [23], via long term
potentiation with ionotropic and metabotropic glutamate
receptors. During activation of glutamatergic synapse in
pyramidal neurons higher level of glutamate at synaps leads
to long term potentiation via facilitating translocation new
AMPA receptors on synapse [24]. However, in certain
physiological conditions like epilepsy and Alzheimer, ele-
vated extrasynaptic glutamate level serves as a neurotoxic
2511
agent leading to depolarization of neural membranes and to
cell death via extrasynaptic NMDA receptors [25, 26].
Therefore excitotoxicity due to elevated glutamate level
may be a causative factor both for susceptibility to memory
deficit in interictal group as well as progressive memory
deficit with uncontrolled seizures as observed in postictal
group respectively in our study. However reduced glutamate
level in postictal group in comparison to interictal group
observed in our study can be justified with exhaustion of
glutamate after ictal phase as estimation of glutamate was
carried out after cessation of convulsions.
The monoamines (norepinephrine, dopamine and sero-
tonin) represent another group of abundant neuroactive
substances in central nervous system that are capable of
regulating the initiation and spread of seizure activity [27]
and also play vital role in learning and memory formation
[28]. Unanimously, elevated levels of monoamines in the
brain have been speculated to exert an anticonvulsant
activity [29, 30]. However, deficiencies in monoamine
systems are implicated in different types of seizures
including kindling model of experimental epilepsy [31–
34]) possibly via lowering seizure threshold [35]. The
levels of monoamine oxidases, enzymes that catalyze the
oxidation of monoamines in the brain, are reported to be
elevated in platelets in epileptic patients [36, 37] which
may be indirectly responsible for monoamine depletion in
our study. Similarly the elevated monoamine oxidase level
has been reported in patients with Alzheimer’s disease,
most common form of dementia [38].
An altered status of noradrenergic neurons has been
stated in different psychiatric disorders including epilepsy
[27]. Noradrenaline signaling powerfully inhibits seizures,
whereas depletion of norepinephrine increases seizure
susceptibility [39] and accelerates epileptogenesis in vari-
ous animal models [27, 40]. On the other hand, a long
lasting hypothesis also state that adrenergic signaling is
critical for formation of contextual and spatial memory [28,
41]. In our study slight enhancement in noradrenaline level
in postictal group and marked depletion in noradrenaline
level of interictal animals was recorded as compared to
naı̈ve animals. The slight elevation of noradrenaline level
in postictal group of pentylenetetrazole-kindled mice might
be due to activation of the compensatory inhibitory
mechanism of seizures however could not justify associ-
ated learning and memory impairment. Persistent depleted
level of noradrenaline in hippocampus and cortex of
interictal group of pentylenetetrazole-kindled mice can be
well correlated with their lowered seizure threshold [42]
and learning and memory deficit in these animals. Our
observations are in consistent with the previous studies by
Szyndler et al. [43] and Singh et al. [13] regarding the
noradrenaline change in the postictal group and epilepto-
genic foci of epileptic patients [44, 45].
123
2512
The role of dopamine in epilepsy and memory appears
intriguing, complex, and unresolved. Dopaminergic neurons
play important role in the initiation and spread of seizure
activity [29] and helps in consolidation of different forms of
memory by inducing long term potentiation in hippocampal
pyramidal cells [46]. Some report suggests the reduced
dopamine content in epileptic foci of the patients [42, 47]
and might be resulting in inhibition of D1 receptor mediated
GABA release culminating to generalization of seizures
[48]. On the other hand reduced dopamine has also been
reported to cause profound memory loss in primates [49]
possibly via limiting the persistence of D1 receptor mediated
long term potentiation in hippocampus [50]. In our study,
decreased cortical and hipocampal dopamine content in
interictal group of pentylenetetrazole-kindled mice suggests
their susceptibility for learning and memory impairment
while higher degree of cortical and hipocampal dopamine
depletion supports the progressive memory loss in postictal
group. The results of dopamine levels are in line with the
earlier findings of Szyndler et al. [43] however contradicted
by findings of Dazzi and colleagues suggested elevated
extracellular dopamine level in pentyletetrazole-kindled
freely moving mice after 4 days of last seizures [51].
Serotonin, another important monoamine neurotrans-
mitter in central nervous system, is involved in various
pharmacological events by virtue of their diverse receptors
[52]. There is a considerable body of evidence suggesting
serotonergic neurotransmission modulates a wide variety of
experimentally induced seizures [53, 54] and assist mem-
ory formation by potentiating acetylcholine release in
hippocampus via several receptor subtypes (5-HT1A,
5-HT2A/2B/2C and 5-HT4) [55]. Reduction in serotonin level
tends to worsen the seizure severity, possibly by reducing
the seizure threshold [56, 57] and might be leading to
memory deficit. Similarly, in our study marked decrease in
hippocampal serotonin level of postictal group animals
supports the progressive and uncontrolled seizures and
associated memory impairment in these animals. These
findings are consistent with earlier experimental reports
carried out in pentylenetetrazole-kindling [43], electrical
kindling [58] and pilocarpine model of epilepsy [59].
In the central nervous system, nitric oxide could behave
as a secondary and retrograde messenger, neuromodulator,
and neurotransmitter, which may suggest it’s involved in
many physiological (synaptic plasticity and long term
potentiation) and pathological processes [60–63]. Several
evidences demonstrated that excessive production of nitric
oxide causes neurodegeneration culminating to the patho-
genesis of epilepsy, Alzheimer disease and other neuro-
degenerative disorders [63–66]. In our study elevated total
nitrite content in the cortex and hippocampus of pentyle-
netetrazole-kindled mice suggest elevated nitrosative stress
in these areas. This elevated nitrosative stress level can be
123
Neurochem Res (2013) 38:2503–2515
deemed as one of the pathogenic factors for epileptogenesis
and associated learning and memory deficit in pentylene-
tetrazole-kindled mice. However no significant change in
cortical and hippocampal nitrosative stress level in between
postictal and interictal group, suggests no significant dif-
ference in nitrosative stress level with progressive and
uncontrolled seizures.
Hippocampus and cortex are enriched with cholinergic
afferents which, under normal conditions, play a pivotal role
in the control of neuronal excitability [67] and in cognitive
processes [68, 69] and up to some extent modulation of
neuronal excitability which may initiate seizures in epileptic
patients [70, 71]. Chronic pentylenetetrazole treatment in
rodents has been reported to decrease the basal ACh release
in hippocampus which was worsened with pentylenetet-
razole treatment but not with concomitant GABAA agonist
treatment [72]. One of the reasons for decreased basal ACh
level in pentylenetetrazole-kindled mice might be the con-
stitutive overexpression of AChE, linked with elevated
intracellular Ca2? level mediated excitotoxicity and altered
gene expression during seizures. The long lasting changes in
AChE level appease due to splicing of AChE pre-mRNA to
produce unique AChE-R mRNA followed by reduction in
ACh level [67]. The constitutive overexpression of AChE
has been reported to be involved in reduced dendritic
branching and cognitive deterioration [73]. The results of
our study can be advocated in light of above as uncontrolled
seizures in postictal group resulted in increased cortical and
hippocampal acetylcholinesterase activity, suggesting the
progressive memory loss via ACh mediated intracellular
Ca2? augmentation [74] which might be leading to excito-
toxicity and long lasting AChE upregulation as evident in
cortex and hippocampus of interictal group as well.
Although increased ACh level has been reported to improve
memory deficit but in these kindled animals elevated ACh
level might be indulged into ACh dependent excitotoxicity.
The results of the present study suggest reduction in
GABAergic, dopaminergic, serotonergic and enhanced
nitrosative stress and glutamatergic neurotransmission may
play an interesting role in the learning and memory deficit
associated with pentylenetetrazole-kindling. Most of the
AEDs working through GABAergic mechanisms control
seizures, but fail to cure the associated memory deficit in
epileptic patients [9]. Therefore GABAergic approach do
not appears as a probable target for management of
memory deficit in epilepsy. Indeed iGluR receptor antag-
onist will exert anticonvulsant effect [75] but may cause
inhibition of iGluRs dependent LTP formation culminating
to worsening of memory deficit in epileptic patients,
however some studies suggest the use of NMDA antagonist
for the improvement of memory as well [76]. Therefore use
of selective glutamate receptor can be explored further to
evaluate its beneficial effect on learning and memory
Neurochem Res (2013) 38:2503–2515
deficit in epilepsy. Further nitrosative stress levels can be
reduced using NOS inhibitors but their use appears to be
inappropriate due to their controversial role as proconvul-
sant and anticonvulsant [77]. Cholinergic modulation using
acetylcholinesterase inhibitors has long been practiced for
memory complications but it should be cautiously used in
epileptic patients due to their seizurogenic potentials [11].
Therefore use of selective cholinergic modulation in hip-
pocampus and cortex might be useful as adjuvant therapy
for the management of memory deficit in epilepsy. Dopa-
minergic and serotonergic modulation appears suitable as
their elevated level may improves memory and epilepsy.
Therefore use of selective dopaminergic and serotonergic
analogues can be further explored for their effectiveness in
epilepsy as well as memory deficit associated with it.
In conclusion, selective dopaminergic, serotonergic and
glutamtergic facilitation appears to be comprehensive tar-
gets whereas selective cholinergic modulation may be
useful as adjuvant target for the management of memory
deficit in epilepsy. These speculations warrant further
experimental validation, which is underway.
Acknowledgments The authors would like to acknowledge the
Indian Council of Medical Research, New Delhi, India for the grant of
funding for research work and award of Senior Research Fellowship
to Mr. Awanish Mishra (Grant No. 45/33/2010/PHA-BMS) and
Department of Pharmaceutical Sciences and Drug Research, Punjabi
University, Patiala to provide infrastructures and other facilities to
carry out research work.
Conflict of interest The authors declare that they have no conflict
of interest related to this manuscript.
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