Effect of Intrahippocampal Injection of Aluminum on Active Avoidance Learning in Adult Male Rats
S. Zahedi Asl
Aim of this research was to study the effect of intrahippocampal
injection of different doses of AlCl3 in adult male rats on
active avoidance learning. Thirty five adult male Wistar rats (250-300
g) were used into five groups: (1) Control, (2) Test-I received daily
1 μL AlCl3 1%, pH = 7.2, 3); Test-II received daily 1
μL AlCl3 0.5%, pH = 3.4, 4); Sham-I received daily 1 μL
aCSF, pH = 7.2, 5); Sham-II received daily 1 μL aCSF, pH = 3.4. All
rats in test and sham groups treated 10 min before training. Animals were
anaesthetized with ketamine HCl/xylazine (90/10 mg kg-1 b.wt-1,
i.p.) and underwent a stereotaxic surgery for implant of two stainless
steel guide cannula into the hippocampus bilaterally. Every day 10 min
after above treatments all rats were used to assess the spatial learning
performing using Y-maze. Criterion Correct Response (CCR) was 90% in last
session of training. There were no significant differences between training
sessions to receiving CCR in control, Sham-I and Sham-II groups. Cognition
in animals received AlCl3 1%, pH = 7.2 was impaired significantly
with compare to other groups (*p<0.0001). Present results show that
intrahippocampal injection of AlCl3 1%, causes active avoidance
learning impairment significantly. The exact mechanism of Al3
effect on brain and cognition is remains unknown.
Aluminum (Al3), a known neurotoxin, causes extensive damage to the
nervous system, including the impairment of learning and memory. Chronic Al3-exposure
in rats is associated with neuronal apoptosis in brain and impaired learning
and memory (Niu et al., 2007). Aluminum salts
are added to a range of commercially-prepared foods and beverages: to clarify
drinking water, make salt free-pouring, color snack/dessert foods and make baked
goods rise (Walton, 2007). AlCl3 in drinking
water for 8 months causes deficits in rat spatial learning and memory (Luo
et al., 2007). Investigators have suggested that learning and memory
deficit of rats could be induced by AlCl3 solution and acethylcholinestrase
(AChE) expressions in hippocampus were increased (Gong et
al., 2006). Studies have shown that Wistar rats were given daily aluminum
chloride 500 mg kg-1, i.g, for one month, followed by continuous
exposure via the drinking water containing 1600 ppm aluminum chloride for up
to 5 months, significantly increased escape latency and searching distance when
tested by Morris water maze, indicative of brain dysfunction (Gong
et al., 2005). In the other hand, aluminum-induced learning and memory
impairment model was established by gavage of aluminum chloride (600 mg kg-1)
for 3 months (Shi-Lei et al., 2005). In compare
with controls, the synapses in aluminum-induced rats exhibited significant changes
such as decreased thickness of postsynaptic density (PSD), increased width of
synaptic cleft, increased numbers of flat synapse, decreased numbers of positive
curvature synapse and perforated synapse and significantly increased aluminum
deposits in hippocampus and frontal cortex. These findings indicate that aluminum
can decrease the ability of rats to learning and memory and induce their synaptic
configuration changes, that may be related to synaptic efficacy and may be one
of the mechanisms for Al3 to induce Alzheimer`s Disease (AD) (Jing
et al., 2004). Excess aluminum exposure impairs neurocognitive in
humans and animals.
Epidemiological studies have shown a potential link between chronic Al3
exposure and Alzheimer`s disease. So, aluminum has been etiologically and epidemiologically
related to several neurologic conditions, including AD (Zhang
et al., 2003). Al3 overload caused significantly increased
level of Al3 in serum. Brains of experimental animals, studied by
optical microscopy, displayed a massive cellular depletion in the hippocampal
formation, particularly, the CAl field and also in the temporal and
parietal cortex. These behavioral and neuropathological modifications associated
with long-term exposure to Al3 are reminiscent of those observed
in AD (Miu et al., 2003). After exposure young
and old male rats to 100 mg/kg/day of Al3 as Al3 nitrate
nona hydrate in drinking water concurrently with citric acid (356 mg/kg/day)
for a period of 100 days, there were no significant effects of Al3
exposure between groups could be detected on behavior, while the total number
of synapses decreased with age and Al3 exposure (Colomina
et al., 2002).
Although the neurotoxic actions of aluminium have been well documented,
its contribution to cognitive impairment such as avoidance learning and
memory remains controversial. In this study the effect of intrahippocampal
bilateral administration of different doses of AlCl3 in adult
male Wistar rats on active avoidance learning in equal 3-arms Y-maze was
MATERIALS AND METHODS
Animals: Thirty five adult male Wistar rats (250-300 g) were used
as subjects in the present experiment (from Lab Animal Care and Breeding
Center of Ahwaz Jondishapour University of Medical Sciences AJUMS, Iran).
This study was conducted from 25th December of 2006 to 20th December of
2007. All animals were housed individually per cage under a 12 h light/dark
cycle, 20 ± 2°C temperature and 60-65% humidity controlled
room with food and water ad libitum. All procedures were approved
by the Institute Research Ethics, Animal Care and Use Committee of AJUMS.
Rats were divided randomly into five groups 7 in each: (I) Control without
any surgery or Al3 administration, (II) Test-I received 1 μL
AlCl3 1% and pH 7.2, (III) Test-II received 1 μL AlCl3
0.5% and pH 3.4, (IV) Sham-I received 1 μL aCSF and pH 7.2, in order
to evaluate the effect of injected volume on learning, (V) Sham-II received
1 μL aCSF and pH 3.4, in order to evaluate the effect of acidic pH
on learning. All animals received drugs into hippocampi bilaterally daily
10 min before training.
All rats in test and sham groups were anaesthetised with ketamine hydrochloride/xylazine
(90/10 mg kg-1, i.p.) and underwent a stereotaxic surgery. In groups
undergoing microinjection, two stainless steel guide cannula (0.6 mm, O.D.)
with an inner needle guide (0.3 mm, O.D.) were inserted into the hippocampus
bilaterally at stereotaxic coordinates: P, 2.2 from bregma, L, ± 2; H, 3 mm
from skull surface (Paxinos and Watson, 1986). All implants
were fixed to the skull with acrylic dental cement and two anchor screws. All
injections were done 7-10 days post surgery recovery. Drugs were injected at
the rate of 1 μL min-1. The needle remained in place for an additional
5 min following the infusion and then it was slowly withdrawn. The animals in
the sham groups were injected with an equivalent dose of aCSF with the same
Training procedure: Every day 10 min before the above treatments, the
five groups of rats were used to assess the spatial learning performing using
an equal 3-arms Y-maze with using an A/D converter, a special software on a
PC as active avoidance learning as reported previously (Sarkaki
and Karami, 2004). The device is composed of three arms (of equal length,
separated from each other at an angle of 120 degrees), with an array of stainless
steel rods on the floor of the arms through which an electric current can be
applied. Each arm has a lamp on the end top and the electric power and the lamp
can be turned on individually when needed. Training was done as one session,
30 trials daily. Animals were conditioned using a 15 watts light as Conditioned
Stimulus (CS) and 20-25 V electrical foot shock (AC, 150-200 mA, 50 Hz, 200
μsec pulse wide) as unconditioned stimulus (UCS). Inter-Trials Interval (ITI)
and Inter-Stimuli Interval (ISI) were 60 and 5 sec, respectively. Trained animals
left the dark arms and enter in light arm during 5 sec delay time (ISI). This
effort was counted as conditioned response. Criterion Correct Responses (CCR)
were 90% in last session of training. Training session number was same for control,
test and sham groups.
Statistical analysis: The data of percents of correct responses
in each group presented as Mean ± SEM were analyzed for significant
differences by one-way ANOVA followed by Tukey`s post hoc test. A p-value
less than 0.05 were assumed to denote a significant difference and level
of significance is indicated by asterisk: *p<0.0001.
Data showed that training sessions in control group to receiving 90%
correct responses (Criterion Correct Responses or CCR) of 30 trials per
session daily was 5.33 ± 0.56. Training sessions to CCR in Sham-I
group that received intrahippocampal 1 μL aCSF, pH = 7.2 bilaterally
was 5.5 ± 0.43. In other hand mean training sessions to CCR in
Sham-II group that received intrahippocampal 1 μL aCSF, pH = 3.4
bilaterally was 6 ± 0.45. There were no significant differences
between mean training sessions to receiving CCR level in control with
both Sham-I and Sham-II groups (Fig. 1).
||Percents of criterion correct responses of control, aCSF with
pH = 7.2 and aCSF with pH = 3.4 groups during training sessions in Y-maze
||Percents of criterion correct responses of control, AlCl3
with pH = 7.2 and AlCl3 with pH = 3.4 groups during training
sessions in Y-maze. CCR% was reduced significantly in group receiving AlCl3
pH =7.2 (*p<0.0001)
Mean training sessions to receiving CCR in test-I group that treated
with intrahippocampal 1 μL AlCl3, pH = 3.4 bilaterally
was 5 ± 0.52 and did not different with control group significantly.
So, acidic pH solutions (pH = 3.4) such as aCSF or AlCl3 could
not affect mean training sessions to receiving CCR. Mean training sessions
to receiving CCR in test-II that treated with intrahippocampal 1 μL
AlCl3, 1%, pH = 7.2 bilaterally was different significantly
from third training session (p<0.0001) in compare to control and all
other groups. While animals in test-II group trained extra three sessions
could not receive to CCR level (Fig. 2), other groups
usually trained 6 to 7 sessions to receiving CCR.
||Comparison of criterion correct responses percents in control,
aCSF with pH = 7.2, aCSF with pH = 3.4, AlCl3 with pH =7.2 and
AlCl3 with pH = 3.4 groups during training sessions in Y-maze.
CCR% was reduced significantly only in group receiving AlCl3 pH
= 7.2 (*p<0.0001)
As it appears animals in two groups including control and sham-1(aCSF
with pH = 7.2) received to CCR after seven training sessions, while animals
in group test-1 (0.5% AlCl3, pH = 3.4) received to CCR one
session later (Fig. 2).
But cognition in animals that received AlCl3 1%, pH = 7.2
was impaired significantly with compare to other groups (p<0.0001).
So, central administration of AlCl3 could impair cognitive
function as dose-dependent (Fig. 3).
Results of this research show that intrahippocampal injection of 1% AlCl3
impairs active avoidance learning significantly.
Intraperitoneal injection of AlCl3 for 60 days in Sprague-Dawley
rats could decrease active avoidance response and spontaneous motor activity
in the shuttle-box test and the open field test significantly. Granulovacuolar
degeneration (GVD) of nerve cells in hippocampus was observed and the number
of GVD cells increased significantly. The incidence of GVD per 300 nerve cells
was significantly related to the dosage of aluminum (Sun
et al., 1999). Intracerebroventricular (ICV) injection of aluminum
tartrate produces transient regional cerebral glucose uptake (rCGlu) depression
in caudate-putamen, geniculate bodies and periaquaeductal gray (Provan
and Yokel, 1992).
Al3, after 4 weeks administration, had a deleterious effect on the
activities of biosynthetic (choline acetyltransferase) and hydrolytic (acetylcholinesterase)
enzymes of the neurotransmitter acetylcholine. The levels of acetylcholine were
also significantly lowered in different brain regions at the end of the dose
regimen. There was a significant decrease in high-affinity choline uptake following
Al3 exposure and number of binding sites of muscarinic acetylcholine
receptor decreased with the maximum effects being manifested in the hippocampus.
The impaired cholinergic functioning had severe effects on cognitive functions.
These results suggest that Al3 exerts its toxic effects by altering
cholinergic transmission, which is ultimately reflected in neurobehavioral deficits
(Julka et al., 1995).
Although Al3 contributes to a variety of cognitive dysfunctions
and mental diseases, the underlying mechanisms of Al3 interactions
with the nervous system are still unknown. The negative action of Al3
on synaptic transmission and Long-Term Potentiation (LTP) by performing electrophysiological
recordings both in vivo, using freely moving animals and in vitro,
using hippocampal slices was confirmed (Platt et al.,
1995). Studies on the effect of aluminum on the brain of rats exposed to
this metal (500 mg Al3 L-1 in drinking water) daily for
180 days showed significant reduction in the spontaneous locomotor activity
was noticed after 90 and 180 days of Al3 exposure to the rats, the
magnitude of the change being almost identical at both the time intervals. Aluminum
exposure also produced significant deficits in acquisition and retention of
learned response in rats, these changes being time dependent. They indicated
significant increase in the lipid peroxidation and decrease in the activity
of Mg2+-ATPase and Na+, K+-ATPase in the brain
of rats. Al3 may is responsible to initiate neurotoxic effects by
producing changes in the structure and function of the plasma membrane needs
further investigations (Lal et al., 1993).
Investigators tested the hypothesis that Al3-induced inhibition
of learning may be due to its effect on glutamate release secondary to changes
in calcium channel function and/or intracellular events triggering glutamate
release. It is suggesting an Al3-induced alteration of Ca2+channel
function. These effects were prevented by the Gi protein inhibitor N-ethylmaleimide,
suggesting an effect of Al3 on the Gi protein to inhibit glutamate
release. Suggesting an Al3 modulation of protein kinase C (PKC)-evoked
glutamate release. These results demonstrate an Al3 inhibition of
glutamate release that may be mediated by multiple, but interconnected mechanisms
(e.g., via interactions with Ca2+ systems), providing multiple targets
for an Al3-induced alteration of neuronal function (Provan
and Yokel, 1992).
It was found that after long-term exposure to Al3, it was concentrated
in white matter of the medial striatum, corpus callosum and cingulate bundle
and the spontaneous motor ability in the open field and the latency of passive
avoidance in aluminum treated rats were decreased as compared with the controls.
In hippocampus, the contents of aspartate (Asp) and glutamine (Gln) were significantly
decreased while taurine (Tau) was significantly increased at higher doses of
Al3 as compared with controls. The altered content of amino acid
neurotransmitters in hippocampus might be one of the important mechanisms of
aluminum neurotoxicity (Jia et al., 2001). Damage
of the cingulate bundle in Al3-treated animals led to a severe anterograde
degeneration of cholinergic terminals in cortex and hippocampus, as indicated
by acetylcholinesterase labelling. It was suggested that the enhancement of
inflammation and the interference with cholinergic projections may be the modes
of action through which Al3 may cause learning and memory deficits
and contribute to pathological processes in AD (Platt et
Intracerebroventricular (ICV) microinjection of aluminum (5.0 μg in 2.0 μL),
once a day for 5 days could cerebral damage. Using meloxicam, a selective inhibitor
of cyclooxygenase-2 (COX-2), has putative protective effects on the oxidative
damage induced by aluminum overload in mouse brain. These evidences approved
impairment of learning and memory function was caused by aluminum overload (Jun-Qing
et al., 2006).
According to the World Health Organization, oral ingestion of aluminum additives
is the main form of aluminum exposure for the general public. Wistar rats that
chronically consumed aluminum in an amount (1.5 mg kg-1 b.wt.) equivalent
to the high end of the total aluminum range ingested daily by humans living
in contemporary urban society, their hippocampal neurons stained for aluminum,
showing some but not all features of aluminum accumulation that occur in human
hippocampal neurons. In view of evidenced linkages of aluminum with beta-amyloid
plaque and neurofibrillary tangle formation in humans with Alzheimer`s disease,
the findings suggest this protocol is worsting in larger groups of animals (Walton,
Compared with the rats in the control group, the learning and memory abilities
of the Al3-exposed rats were significantly decreased. The content
of MDA was increased while the activity of SOD was decreased. The membrane structure
of neurons in cerebrum cortex of the Al3-exposed rats were broken,
dissolved and gone. Aluminum can accelerate lipid peroxidation in rat`s brain,
which may be one of the important intoxication mechanisms of aluminum (Zhang
and Yu, 2002; Yang et al., 2006; Li
et al., 2006; Kaneko et al., 2006).
An investigation on possible effects of chronic aluminium exposure on neurofilament
phosphorylation and its subsequent disruption in various regions of the rat
brain showed that an intra-gastric dose of aluminium (10 mg kg-1
b.wt. for 12 weeks) resulted in a marked enhancement of Ca2+/CaM
dependent protein kinase activity as compared to cAMP dependent protein kinase.
The levels of phosphoprotein phosphatase were found to be significantly depleted
only in the cerebral cortex. The cytoskeletal proteins were found to be aggregated
and disrupted neuronal regions following 12 weeks of aluminium treatment. This
study lends further support to the possible role of aluminium as a potent neurotoxic
agent and in the etiopathogenisis of various neurodegenerative diseases (Kaur
et al., 2006).
Cognition in animals that received AlCl3 1%, pH = 7.2 was
impaired significantly with compare to other groups (p<0.0001) due
to Al3 accumulation in brain tissue. So, everybody needs to
know that any contact with aluminum (beverages, foods, industrial and
etc.) may act as toxic and impairs cognitive function as dose-dependent.
Support for this research (MU. 82) was provided by funding from Physiology
Research Center, Ahwaz Jondishapour University of Medical Sciences (AJUMS).
The authors also thanks AJUMS animal house experienced personnel.
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