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Introduction
Epilepsy is frequently accompanied by impairments in
various cognitive functions. More than 45% of epileptics
have psychological or social problems with behavioral
manifestations and are completely or partly
disabled[1]. Although the causes of cognitive impairment in patients with epilepsy
have not yet been completely elucidated, 3 factors are
proposed: the underlying etiology of the epilepsy, the
effects of seizures themselves, and the side-effects of
antiepileptic drugs (AEDs) at therapeutic
doses[2]. There is a need for drugs or adjuvants that can simultaneously
suppress seizures and ameliorate the concomitant cognitive
impairment.
Brain histamine levels might play an important role in the
regulation of seizure susceptibility. Injection of histidine ip,
a precursor of histamine, and metoprine, a histamine
N-methyltransferase inhibitor, or icv injection of histamine,
inhibited seizures induced by pentylenetetrazol, maximal
electroshock (MES) or amygdaloid kindling in mice or
rats[3-6], whereas a-fluoromethylhistidine, a selective and
irreversible histidine decarboxylase inhibitor, increases the duration
of clonic convulsions induced by MES in
mice[3] and causes severe seizure development in pentylenetetrazol-induced
kindling[7]. In contrast, the involvement of histamine in
learning and memory processes has also been well documented.
Histamine significantly ameliorates memory deficits induced
by aging, dorsal hippocampal lesions, scopolamine and
MK-801, as determined in rats using passive and active
avoidance tasks and the 8-arm radial
maze[8-12]. It is proposed that histamine and histaminergic compounds might be useful
adjuncts for conventional AEDs, with dual advantages: one
for enhancement of the anticonvulsant efficacy of AEDs,
and the other for improvement of the memory deficits
occurring with epilepsy.
The objectives of the present study were to further clarify
whether histidine can both enhance the anticonvulsant
efficacy of AEDs and ameliorate the spatial memory impairment
induced by seizures. The chronic transauricular kindling
procedure was used in rats as a new animal model of epilepsy,
and an 8-arm (4 arms baited) radial maze paradigm was used
to evaluate spatial memory and differentiate between
short-term and long-term memory.
Materials and methods
Animals All experiments were carried out in accordance
with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals. Male Sprague-Dawley rats (220-270 g, Grade II, Certificate No 22-9601018, Experimental
Animal Center, Zhejiang University) were maintained in an
air-conditioned room at 22-26 °C and 40%-70% humidity, and
housed in individual cages with a 12-h light-dark cycle (lights
on from 8:00 to 20:00). Animals were given free access to
water and kept at 80%-85% of their free feeding body weight
throughout the radial-arm maze experiments. Experiments
were carried out each day between 10:00 and 17:00.
Radial-arm maze training The apparatus used is
described in our previous
reports[9,10,13]. The rats were
familiarized with the radial maze once per day for 2 d prior to training.
Food pellets (45 mg each, Bio-Serv) were scattered over the
entire maze surface, and 3 or 4 rats were simultaneously
placed in the maze and allowed to explore and take food
freely for 10 min. After adaptation, all rats were trained with
1 trial per day. In each trial, only 4 arms (3, 5, 6, and 8) were
baited, and the sequence was never changed throughout
the experiment. One rat was placed on the center platform
that was closed off by a door. After 15 s, the door was
opened and the rat was allowed to make an arm choice to
obtain food pellets until all 4 pellets had been eaten or 5 min
had elapsed. The number of entries into unbaited arms was
regarded as the total error (TE). The number of entries into
never-baited arm was taken as reference memory error (RME),
whereas re-entry into arms where the pellet had already been
eaten was considered as a working memory error (WME).
Rats continued training until reaching a criterion of less than
1 error per trial for 5 consecutive trials. Memory retrieval
was tested in the same maze.
Chronic transauricular kindling After successful
training in the radial maze, each rat in the experimental group was
given 1 subconvulsive electrical stimulation daily via
ear-clip electrodes (40 mA, 0.2 s, Hugo Saches type 221) until
fully kindled. Animals in the sham group had the electrodes
applied, but no current was delivered. Each rat was placed
separately under a glass funnel, and the appearance of
clonic-tonic seizures was recorded. When rats exhibited
clonic-tonic seizures after each of 3 consecutive stimulations, they
were regarded as fully kindled and used for the drug study.
The endpoint of efficacy was taken as the inhibition of tonic
hindlimb extension (HLE). The number of animals showing
complete abolition of tonic HLE was expressed as percent
protection.
Measurements of brain histamine and g-aminobutyric
acid (GABA) content
Sample preparation Rats were sacrificed by
decapita-tion. The brain was quickly removed, placed on an ice-cold
stainless steel plate, and dissected into the cortex,
hippo-campus, brainstem and hypothalamus according to the meth
ods of Glowinski and Iversen[14]. The brain tissues were
stored at -80 ºC until assayed. The tissue was homogenized
in 3% perchloric acid containing 5 mmol/L disodium EDTA
and 5-hydro-Nw-methyltryptamine in a Polytron homogenizer
(Kinematica) at the maximum setting for 20 s in an ice bath.
The homogenate was centrifuged at 15
000×g at 4 ºC for 20 min. Then, the supernatant was removed and filtered
with a 0.22 µm polyvinylidene difluoride membrane.
Chromatographic conditions Tissue samples were
analyzed by high performance liquid chromatography (HPLC)
combined with electrochemical detection using a technique
developed in our laboratory for the simultaneous and
sensitive analysis of histamine and GABA. The system consisted
of a model 582 pump, a model 540 autosampler and a
4-channel CoulArray electrochemical detector. The HPLC was
controlled and the data acquired and analyzed using CoulArray
software. All of the above equipment was obtained from
ESA. After reacting with the derivate o-phthalaldehyde,
analytes were separated on a 3 mm, 3 mm×50 mm Capcell Pak
MG C18 column from Shiseido. A 2-component gradient
elution system was used, with component A of the mobile phase
being 100 mmol/L
Na2HPO4, 13% acetonitrile, and 22%
methanol, pH 6.8, and component B being similar to A except
with 5.6% acetonitrile and 9.4% methanol. The gradient
elution profile was as follows: 0-3.5 min, isocratic 100% B;
3.5-20 min, linear ramp to 0% B; 20-22 min, isocratic 0% B;
22-23 min, linear ramp to 100% B; 23-30 min, isocratic 100% B.
Flow rate was set to 0.75 mL/min. The first cell was set
at +250 mV, whereas the second cell was set at +350 mV. All
standards were obtained from Sigma. The retention times of
GABA and histamine were 15.16 min and 18.36 min,
respectively.
Drugs Histidine monohydrochloride and carbamazepine
(CBZ) were obtained from Sigma. Histidine was dissolved in
saline, and CBZ was suspended in a 1% solution of Tween
80. All drugs were injected ip in a volume of 1 mL/kg of body
weight. Histidine was injected 1 h before and CBZ was
injected 0.5 h before transauricular electrical stimulation or
before the radial maze test. Drugs were given once a week.
The same animals were used repeatedly, and they
experienced all doses of each drug given in ascending order.
Statistical analysis Results were expressed as mean±
standard error of the mean and analyzed by one-way
analysis of variance followed by DunnettĄ¯s
t-test. For percentage incidence, the
c2-test with FisherĄ¯s exact test was used. All
statistical analyses were carried out using SPSS 11.5 for
Windows. P<0.05 was considered as statistically significant.
Results
Effects of histidine and CBZ on transauricular kindled
seizures in rats Both histidine and CBZ inhibited
transauricular kindled seizures in a dose-dependent manner,
as reflected by the increase in percentage protection against
tonic HLE. Histidine at doses of 200 mg/kg and 500 mg/kg
slightly inhibited tonic HLE, but had no significant effect,
whereas at doses of 1000 mg/kg and 1500 mg/kg, it
significantly inhibited tonic HLE (P<0.05). CBZ at doses of 1
mg/kg and 2 mg/kg showed a tendency to inhibit tonic HLE,
but no significant effect was observed, whereas at doses of
5 mg/kg and 10 mg/kg, it provided significant protection
against tonic HLE (P<0.01). In addition, histidine at a low
dose of 200 mg/kg showed a tendency to potentiate the
anticonvulsant effects of 1 mg/kg CBZ and it significantly
potentiated the anticonvulsant effects of 2 mg/kg CBZ
(P<0.05). At a dose of 500 mg/kg histidine significantly
potentiated the anticonvulsant effects of CBZ (1 mg/kg or 2 mg/kg)
(P<0.05 and P<0.01, respectively) (Figure 1).
Effects of histidine and CBZ on radial maze performance
in rats with spatial memory deficits induced by chronic
transauricular kindled seizures The chronic transauricular
kindling resulted in a significant increase in the number of
TE, WME, and RME in the spatial memory retrieval process
(P<0.01). Histidine at doses of 200 mg/kg and 500 mg/kg
significantly decreased the number of TE and WME
(P<0.05), but not the number of RME. However, histidine at
doses of 1000 mg/kg and 1500 mg/kg had no ameliorative
effect on the spatial memory deficits induced by transauricular kindled seizures. In contrast, CBZ (2, 5, 10,
and 20 mg/kg) had no effect on memory impairment. The
co-administration of CBZ also did not influence the
improvement of spatial memory deficits induced by 500 mg/kg
histidine (Table 1).
Effects of histidine and CBZ on brain histamine content
Compared with control rats, chronic transauricular kindling
produced a significant decrease in the histamine content of
the hypothalamus (41.8%), hippocampus (43.6%), and
brainstem (57.3%) (P<0.05). Histidine (500 mg/kg, ip)
significantly increased the histamine content of the hypothalamus
(157.9%) (P<0.05) and slightly increased that of the cortex
(114.7%), hippocampus (112.7%) and brainstem (122.7%).
CBZ (2 mg/kg) had no effect on the increased histamine
content induced by histidine treatment (Table 2).
Effect of histidine and CBZ on brain GABA
content Neither treatment with histidine (500 mg/kg) or CBZ (2
mg/kg) alone, nor co-administration of the 2 drugs had any
effect on brain GABA levels (Table 3).
Discussion
In the present study, we provide evidence for the first
time that chronic transauricular kindling can induce not only
generalized tonic-clonic seizures but also spatial memory
deficits in rats. So far, few animal models are suitable for
studing the memory deficits associated with epilepsy,
especially for the form with generalized tonic-clonic
seizures[15]. MES is a representative animal model of generalized
tonic-clonic seizures[16]; however, it has no appreciable effect on
cognitive behaviors, such as in passive avoidance
learning[17,18]. This might be because MES is a model of acute
seizures (reactive or provoked), rather than a model of
epilepsy[19]. In the present study, we developed a chronic
transauricular kindling model that was relatively simple and
required neither expensive equipment nor highly skilled
personnel. We found that chronic transauricular kindled
seizures produced spatial memory deficits, which remained
in a steady state for approximately 3 weeks after fully kindled
(data not shown). Therefore, it is likely that the chronic
transauricular kindling model is a very useful animal model
for evaluating memory deficits associated with epilepsy, and
in drug screening for both anticonvulsant and
memory-improving actions.
In the present study, high doses of histidine (1000 mg/kg
and 1500 mg/kg), a precursor of histamine, significantly
inhibited chronic transauricular kindled seizures, but had no
ameliorative effect on the spatial memory deficits induced
by these seizures in rats. However, at doses of 200 mg/kg
and 500 mg/kg, although it had no appreciable
anticonvulsant effect, histidine significantly ameliorated spatial memory
deficits. Therefore, it is proposed that at a lower dose,
histidine combined with CBZ can simultaneously enhance the
anticonvulsant effects and ameliorate spatial memory
impairment in rats. Interestingly, we found that the lower doses of
histidine significantly potentiated the protective effects of
CBZ against transauricular kindled seizures and the
cognitive improvement was not affected by co-administration of
CBZ. Therefore, our results indicate that histidine, at a
specific dosage range, potentiates both the anticonvulsant
efficacy of CBZ and ameliorates the spatial memory deficits
induced by chronic transauricular kindled seizures in rats. This
indicates that histidine might serve as a beneficial adjuvant
for the clinical treatment of epilepsy.
In addition, we found that chronic transauricular kindling
resulted in a marked decrease of histamine content in the
hypothalamus (41.8%), hippocampus (43.6%) and brainstem
(57.3%). Consistent with our findings, decreased histamine
content of the amygdala and hypothalamus has been
reported to follow amygdaloid
kindling[20]. The observed decrease in histamine levels following chronic transauricular
kindled seizures further supports the concept of histamine
as an endogenous anticonvulsant[21]. However, in contrast
to our data, Vohora et al reported that acute MES
significantly increased histamine concentration in the
brainstem[22]. We have no data to explain this difference, which might be a
result of different mechanisms underlying the seizures (acute
vs chronic). Therefore, our results might at least partly
elucidate why acute MES has no appreciable effect on cognitive
behavior, whereas chronic transauricular kindling induces
significant spatial memory deficits in
rats[17,18].
It remains unclear why and how histidine potentiates the
anticonvulsant effect of CBZ. A possible pharmacokinetic
interaction is unlikely, because the plasma levels of CBZ
remain unchanged in the presence of
histidine[17]. Further-more, we found the GABA levels in the brain after treatment
with histidine and/or CBZ were not significantly different
from the values in the control group, which suggests that
the enhancement of the anticonvulsant effect of CBZ at the
doses used is independent of GABA levels. However,
treatment with histidine (200 mg/kg or 500 mg/kg) significantly
increased brain histamine content in the hypothalamus. The
significant increase of brain histamine content induced by
injection of histidine might at least partly contribute to the
observed enhanced anticonvulsant efficacy of CBZ.
It is interesting that the effects of histidine on
performance in the radial maze showed a bell-shaped inhibition.
These results are in accordance with the reports by Sakai
et al[23] and Ghi
et al[24], who found dose-dependent biphasic
effects of histamine on locomotor activity. Alvarez
et al also reported that treatment with a lower dose of histamine
(45 nmol) in the hippocampus improved memory retrieval in
contrast to the effect of a higher dose
(90 mg/kg)[25]. These findings suggest that the biphasic effects of histamine in the
brain might be mediated through different mechanisms, and
the memory improvement induced by histidine only occurs
within a specific lower range of dosage.
We were further interested to find that histidine
significantly decreased both the number of TE and WME, but not
the number of RME induced by transauricular kindled
seizures in rats. Our data suggests that histidine can only
ameliorate the short-term memory deficit induced by the kindled
seizures. Similar findings show that histidine, at doses that
cause a significant increase of brain histamine content,
improves the working memory deficit induced by 8-OH-DPAT
and 7-chlorokynurenic acid in
rats[26,27]. Our previous work also shows that the
H3-antagonist clobenpropit facilitates deficits of working memory induced by intrahippocampal
application of MK-801, and we demonstrate that the effects
on working memory are a result of an increase in endog
enous histamine, whereas the effects on reference memory
are most likely a result of neurotransmitters other than
histamine[28]. From the above evidence, it is likely that different
mechanisms underlie working memory and reference memory,
and brain histamine mainly participates in the former.
Because short-term memory complaints are frequent in patients
with epilepsy[29], the improvement in working memory
induced by co-treatment with CBZ and histidine appears
potentially useful.
In conclusion, we found that chronic transauricular
kindling in rats was a very useful animal model for evaluating
memory deficits associated with epilepsy. Histidine at
relatively low doses significantly enhanced the anticonvulsant
efficacy of CBZ against the kindled seizures and also had a
significant ameliorative effect on the spatial memory
impairment induced by these seizures. It is proposed that histidine,
at a specific range of dosages, might serve as a beneficial
adjuvant for the clinical treatment of epilepsy, especially
when it is accompanied by impairment of spatial memory.
Acknowledgement
We are very grateful to Dr Iain C BRUCE (University of
Hong Kong) for reading the manuscript.
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