NeuroToxicology 29 (2008) 1069–1079

Aluminium-induced electrophysiological, biochemical and cognitivemodifications in the hippocampus of aging rats

Pallavi Sethi a,b, Amar Jyoti a, Rameshwar Singh a, Ejaz Hussain b, Deepak Sharma a,*a Neurobiology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, Indiab Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India

A R T I C L E I N F O

Article history:

Received 7 March 2008

Accepted 22 August 2008

Available online 5 September 2008

Keywords:

Multiple unit activity

Aluminium chloride

Protein kinase C

Electrophysiology

Morris water maze

Hippocampus

A B S T R A C T

Aluminium (Al) is the most abundant metal known for its neurotoxicity in humans. It gains easy access to

the central nervous system under normal physiological conditions and accumulates in different brain

regions. It has been reported to be involved in the etiology of several neurodegenerative diseases. In this

study, we have investigated the effects of long-term intake of aluminium chloride (AlCl3) on the

electrophysiological, behavioral, biochemical and histochemical functions of hippocampus. Wistar rats

were fed with AlCl3 at a dose of 50 mg/(kg day) for 6 months in the drinking water. Effect of long-term

intake of Al was studied on the electrical activity of hippocampal CA1 and CA3 regions in brain of young

and old rats. Morris water maze and open field tests were performed to investigate the cognitive and

anxiety status of aging rats intoxicated with aluminium. Our studies indicate that aluminium intake

results in increased multiple unit activity and adversely affect the spatial learning and memory abilities

of both young and old rats. Aluminium intake also inflicts oxidative stress-related damage to lipids,

membrane associated proteins (Na–K ATPase and PKC) and endogenous antioxidant enzyme activity

(SOD, GPx and GST). The compromised antioxidant system might be playing a crucial role in the observed

Al-induced alterations. We have observed that the magnitude of AlCl3-induced alteration was

considerably higher in younger group of rats compared to older group. In conclusion, the results of

the present study implicates that aluminium treatment exerts its neurotoxic effects by altering the

overall physiology of brain, and the induced changes were strongly correlated with each other.

� 2008 Elsevier Inc. All rights reserved.

Contents lists available at ScienceDirect

NeuroToxicology

1. Introduction

Aluminium (Al) is the third most abundant element on the earthcrust and gets an easy access to our body through use of cookingutensils, deodorants, antacids, etc. (Yokel, 2000). Al is routinely usedas a water treatment reagent and is often added in the processing offood and pharmaceutical products (antacids). Therefore, our dailyintake of aluminium ranges from 10 to 20 mg/kg via digestive andrespiratory tracts (Edwardson et al., 1992). Epidemiological studieshave also reported that increased Al in drinking water is associatedwith geographical prevalence of Alzheimer’s disease (Flaten, 2001).Aluminium is reported in the etiology of several neurologicaldisorders like Alzheimer’s disease (Kawahara, 2005), Parkinson’sdisease (Yasui et al., 1992), Guamamian–Parkinsonian complex,amyotrophic lateral sclerosis (Forbes et al., 1995). An increased

* Corresponding author. Tel.: +91 11 26704508; fax: +91 11 26187338.

E-mail addresses: pallavisethiin@yahoo.co.in (P. Sethi),

jyotiamarjnu@yahoo.com (A. Jyoti), ejaz58@yahoo.com (E. Hussain),

dpak57@hotmail.com (D. Sharma).

0161-813X/$ – see front matter � 2008 Elsevier Inc. All rights reserved.

doi:10.1016/j.neuro.2008.08.005

exposure to aluminium alone has not proven sufficient to cause anyof these diseases (except dialysis dementia), the possibility remainsthat elevated level of Al may initiate or aggravate the pathogenesis ofneurodegenerative disease. Aluminium has been reported to alterblood-brain barrier (Banks and Kastin, 1989; Zatta et al., 2002) andgets deposited in the cortex, cingulat bundles, corpus callosum (Plattet al., 2001) and hippocampus (Struys-Ponsar et al., 1997).Aluminium being an inert metal has been suggested to induceoxidative damage indirectly by potentiating the peroxidative effectof Fe2+ (Golub et al., 1999). Aluminium promotes reactive oxygenspecies (ROS) formation when iron is present in its ferrous form(Oteiza et al., 1993). Moreover Al also enhances pro-oxidantproperties of several transition metals like copper and chromium(Bondy et al., 1998). Aluminium administration has been reported toinduce oxidative stress by inflicting damage to membrane lipid,proteins and antioxidative enzyme defense system (Jyoti et al.,2007). In vitro, AlCl3 has been demonstrated to preferentiallyaccumulate in cultured astrocytic cells (Levesque et al., 2000).Reports of in vivo studies suggested that Al-treatment causesapoptosis like changes (Suarez-Fernandez et al., 1999), vacuolatedastrocytes with numerous lipofuscin deposits (Florence et al., 1994),

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–10791070

abnormal mitochondrial swelling, thinning of myelin sheath,cytoplasm with multivesicular bodies (Deloncle et al., 2001) andsynaptic vesicle accumulation (Jyoti and Sharma, 2006). Similarstructural alterations were also reported in normal aging indepen-dent of aluminium intoxication. Therefore, aluminium has beenproposed to accelerate aging process by inflicting oxidative damagein aging related parameters (Deloncle et al., 2001; Kaur et al.,2003a,b). The hippocampus and neocortex are specifically affectedby Al accumulation in the aging humans (Xu et al., 1992).

In the present study, effect of long-term oral Al-administra-tion was assessed at electrophysiological, biochemical andbehavioral levels to investigate the possible pathophysiologyassociated with Al-toxicity. Aluminium induced behavioralalterations as well as cognitive deficit have been widely reportedin literature but exact mechanism is not yet reported. Electro-physiological, biochemical and microscopic studies have beenperformed on hippocampus in order to explain the possiblereason behind behavioral alterations reported and observed inthis study. Neuronal signals were recorded in the form of multipleunit activity (MUA). MUA represents the cumulative neuronalfiring from a group of neurons (Sharma et al., 1993). Hippo-campus was chosen for this study as it is considered to be a keyarea for learning and memory functions (Bliss and Collingridge,1993). In addition, hippocampal neurons participate in encodingas well as retrieval and long-term consolidation of spatialmemory in the Morris water maze (Reidel et al., 1999).Aluminium administration at a dose of 10 mg/(kg day) increasedaccumulation mainly in the hippocampus (�24-fold) followed bycorpus stratum (�5-fold) and cerebral cortex (�4-fold) (Kauret al., 2006). Electrophysiological recordings and histochemicalstudies were performed separately in the CA1 and CA3 fieldof hippocampus as these areas are reported to be involved inboth short-term and long-term memory consolidation (Singh andSharma, 2005).

2. Materials and methods

2.1. Materials

All electrodes and wires used in the electrophysiologicalsurgery were tissue compatible and obtained from PlasticsOne,VA, USA. For biochemical assays, chemicals were obtained fromSigma–Aldrich Chemical Co., Merck, Himedia and SD fine. Allchemicals used were of analytical grade.

2.2. Animals

Forty male Wistar rats of two age groups: young (4 months) andold (18 months) were taken for this study. Rats were housed inpairs, in standard laboratory cages and kept on a 12 h light/12 hdark cycle with ad libitum access to food and water. After surgeryrats were housed individually and continuously monitored fortheir health status. Young and old rats were divided in twosubgroups containing 10 animals in each group (n = 10). Scheme ofgroup formation was as follows: Group 1: Young control, Group 2:Young Al-treated, Group 3: Old control, Group 4: Old Al-treated. Al-treated groups received AlCl3�6H2O at a dose of 50 mg/(kg day) indouble-distilled drinking water for 6 months. Body weight andwater intakes were measured daily to adjust the dose to achieve aconstant intake of aluminium. Same protocol was used to inducealuminium toxicity in our previous publications (Jyoti and Sharma,2006; Jyoti et al., 2007; Kaur et al., 2003a,b). All experimentalprotocols used in the present work were approved by Committeefor the Purpose of Control and Supervision on ExperimentalAnimals (CPCSEA) and animal ethical committee of Jawaharlal

Nehru University, New Delhi, India. Six animals from each groupwere used for biochemical studies and rest (n = 4) were used forhistochemical studies.

2.3. Surgery procedures

Animals were anesthetized using ketamine hydrochloride80 mg/kg and xylazine hydrochloride 10 mg/kg, intraperitoneally,and placed in a stereotaxic apparatus for implantation ofelectrodes. Bipolar wire electrodes were stereotaxically placedin the CA1 and CA3 field of hippocampus as described in the atlas ofPaxinos and Watson (1982) to record EEG and MUA. One screwelectrode was placed upon the frontal sinus to serve as animalground. Insulated (except at the tip) flexible wires were connectedbilaterally to dorsal neck muscles and muscles near externalcanthus of the eyes to record bilateral electromyogram (EMG) andelectrooculogram (EOG), respectively. The free ends of theseelectrodes were soldered to a 15-pin connector, which was fixed tothe skull with dental acrylic to make a robust platform. Operatedrats were provided with optimal post-operative care andhabituation in the recording chamber for 5 days before EEG wasrecorded with the help of Grass polygraph recorder (Model 79D).

EEG was recorded with the help of preamplifier, and signalswere filtered at high cut off 100 Hz and low cut off at 1 Hz. For MUArecordings composite extracellular signals from the same EEGelectrodes were routed through high impedance probe (GrassHIP511 with FET) signals were amplified and filtered (300 Hz to10 kHz) by Grass P511 J preamplifiers, electronically discriminatedand displayed on an oscilloscope. The standard TTL spikes pulsesfrom the window discriminator were simultaneously recorded onthe polygraph. Using grass integrator preamplifier (P10) cumula-tive mathematical integration of EEG traces was recorded on one ofthe polygraph channels. The recordings were limited to the awakeimmobile state in which a rat sits quietly but remain awake asdescribed in previous studies from our lab (Sharma et al., 1993;Kaur et al., 2003a,b).

2.4. Behavioral tasks

Different groups of rats were investigated for their spatiallearning abilities with the help of Morris water maze tests.Ambulation, rearing and anxiety were monitored in the open fieldtests. Scoring procedures are briefly described below.

2.4.1. Open field test

Open field test was performed by modifying the previouslydescribed method by Li et al. (2005). Open field tests wereperformed in a square arena (70 cm � 70 cm � 106 cm) with afloor divided into 49 identical squares of 10 cm length. At thebeginning of the test, the rat was placed in the center of the openfield. Before each trial, the field was cleaned thoroughly with 0.1%acetic acid solution. The locomotor activity (horizontal) defined asnumber of squares crossed and rearing frequency (vertical) definedas number of times the animals stood on their hind legs (Suarez-Fernandez et al., 1999; Colomina et al., 1999) were evaluated.Rearing was recorded manually by the experimenter. Furthermore,defecation index was also counted by counting the number offaecal boles. The number of squares entered with the forepaws bythe rat within a period of 3 min was recorded.

2.4.2. Morris water maze test

Morris water maze task was performed to investigate cue-based learning and memory abilities of rats, following the methoddescribed by Morris (1984) with minor modifications as describedbelow. The maze consisted of a black painted circular tank of

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–1079 1071

168 cm in diameter and 50 cm deep, containing extra maze cueshaving different size, shape and colors. A black circular platform(camouflaged) with an escape of 15 cm diameter was positioned atthe center of one quadrant, which was 2.0 cm under the surface ofthe water so that the rat could escape swimming. Perimeter of thetank was marked at four places pointing north, south, east andwest. Rats selected randomly from the pool were screened for theirswimming abilities, by recording latencies to acquire visibleplatform. Animals were habituated to the experimental conditionsprior to experimentation by placing them on the water tank for60 s without platform (minimum for 4 days) and those exhibitingsignificantly lower swimming speed were discarded for learningand memory test. After 4 days of testing, selected rats were trainedto exit the water tank onto platform by using the visual cues. Eachrat was placed inside the water tank facing the tank wall, at one ofthe four randomly selected entry points once in every block of fourtrials. Eight trials per day were performed, and on each trial thelatency to reach platform was measured. In case an animal fails tofind the platform within 60 s, it was guided to reach the platformand allowed to remain on the platform for 20 s. Each rat wasexposed to the task for four consecutive days (minimum of 20trials). Morris water maze training was recorded using a webcamera mounted to the ceiling (Ozdemir et al., 2005). Recordingwas performed from 11:00 AM to 2:00 PM to exclude theperformance variations resulted due to circadian rhythmicity.

2.5. Biochemical studies

2.5.1. Preparation of tissue homogenate

Rats (n = 6) were killed by cervical dislocation after electro-physiological and behavioral recordings. Brains were quickly takenout and cooled in a deep freezer. Hippocampi were rapidlydissected out on ice plate. The left and right hippocampi of thebrain of one rat were pooled to make one sample of the tissue.Biochemical assays were performed separately in six animals ofeach group. Tissue samples were homogenized in 50 mM Tris (pH7.4) containing protease arrest (Genentech kit) with a Potter-elvehijam type homogenizer fitted with Teflon plunger. Thehomogenate was diluted 1:10 (with Tris, pH 7.4, buffer) andcentrifuged at 6000 rpm for 5 min in a refrigerated centrifuge(Sorvall RCS or RC5C). The resulting pellet (P1), consisting ofnuclear and cellular material, was discarded. The supernatant (S1),containing mitochondria, synaptosomes, microsomes and cytosol,was further ultracentrifuged at 25,000 rpm for 25 min to formmitochondrial pellet (P2). The resulting supernatant (S2) was usedas such as cytosolic fraction. The mitochondrial pellet containsmitochondrial membranes, synaptosomes and microsomes. Thisfraction was considered as membranous fraction (M1).

2.5.2. Estimation of lipid peroxidation and membrane associated

proteins activity

Aliquots of membranous fractions (M1) were used to performthe following assays. TBA-RS content was measured to observelipid peroxidation as described earlier with some modification(Ohkawa et al., 1979). Tetra-methoxy propane (TMP) was used as astandard. Lipid peroxidation was expressed as TBA-RS content permg protein. Na–K ATPase assay was performed as described earlier(Kaur et al., 2003a,b) and iP was estimated as described by Fiskeand Subbarow (1925) with some modification. Ouabain (1 mM)was used as a specific blocker of Na–K ATPase activity. The ouabainsensitive Na–K ATPase activity was estimated and expressed asnmoles of inorganic phosphate released mg protein�1 h�1. Acet-ylcholine esterase (AChE) activity was estimated according to themethod described by Ellman et al. (1961) with minor modifica-tions. The assay mixture contained in 1 ml and final concentration

of following 84 mM sodium phosphate buffer (pH 8.0); 0.32 mMdi-thiobisnitrobenzoate (DTNB) prepared in 0.01 M phosphatebuffer (pH 7.0); 0.48 mM acetylthiocholine iodide as substrate and�150 mg of enzyme protein per assay. The change in OD wasmeasured spectrophotometrically on a UV–vis spectrophotometerat 412 nm. The specific enzyme activity of AChE was calculated asmicromoles of thiocholine produced per min per gram of tissue(mM/(min g)) at room temperature.

PKC assay was performed as described by Hetherington andTrewavas (1982) with some minor modifications. Cytosolic (looselybound) and membrane bound PKC activities were determinedseparately in the cytosolic (S1) and membranous fraction (M1) asdescribed below. The homogenate was suspended in incubationmedium (100 mM HEPES, 120 mM NaCl, 1.2 mM MgSO4, 2.5 mMKCl, 15 mM NaHCO3, 10 mM Glucose, 1 mM EDTA). Protein kinaseactivity was assayed in a total volume of 0.5 ml of incubationmedium [50 mM HEPES (pH 7) buffer, 10 mM MgCl2, 0.5 mM CaCl2,and 0.2 mM EGTA]. In aliquots of 100 mg protein, the reaction wasinitiated by addition of P32 labeled ATP (specific activity, 3000 Ci/mmol ATP). Incubation was carried out at 25 8C. Samples of 50 mlwere taken out at appropriate intervals (60 s) and pipetted onto3 mm filter discs which had been pretreated with 10% trichlor-oacetic acid (TCA), 20 mM sodium pyrophosphate, and 10 mMEDTA. These filter discs were washed in 5% TCA and 10% TCA andextracted in hot ethanol/ether (3:1, v/v) before drying. Radioactivitywas measured by a Beckman-b counter. Results were expressed asb-counts per mg protein/min.

2.5.3. Estimation of antioxidative enzymes

Assays of antioxidant enzymes were undertaken in the aliquotsof cytosolic faction, as cytosolic antioxidative enzymes playimportant roles in cellular antioxidative defense. Superoxidedismutase (SOD) activity was measured according to the methoddescribed by Marklund and Marklund (1974), with some minormodifications. This method is based on the ability of SOD to inhibitthe auto-oxidation of pyrogallol at alkaline pH (8.2). Enzymeactivity was expressed as units/mg protein. One unit is equivalentto the amount of SOD required to inhibit auto-oxidation of 50% ofpyrogallol. The enzyme glutathione peroxidase (GPx) was assayedaccording to the method of Flohe and Gunzler (1984), with somemodifications. The assay takes advantage of concomitant oxidationof reduced nicotinamide dinucleotide phosphate (NADPH) by GR,which is measured at 340 nm. Enzyme activity is expressed asunits/mg protein. The enzyme GST was assayed according to themethod of Habig et al. (1974). The enzyme activity was measuredby following the generation of CDNB-GSH conjugate catalyzed byGST at 340 nm. Protein was estimated in the membranous andcytosolic fraction by the method of Bradford (1976) using bovineserum albumin (BSA) as standard.

2.6. Histochemical studies

Animals (n = 4) were anesthetized and transcardially perfusedfirst with physiological saline and then with a fixative solutioncontaining 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 Mphosphate buffer. For light microscopy, the brain was excised andput in 10% formalin solution. Paraffin sections of thickness 7 mmwere prepared for microscopic study. Finally tissues were dipped inwater and then transferred to cresyl violet stain for 10 min at 60 8C.The stained sections were then washed in running water to removeexcess stain and then upgraded for dehydration through differentgrades of alcohol. Slides were then cleared with xylene and mountedwith DPX to make permanent. The slides were observed under Zeissmicroscope attached with an image acquiring software AxioVision3.0.6. Minimum ten sections through the CA1 and CA3 hippocampal

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–10791072

fields were counted from each animal. The number of neurons in thepyramidal CA1and CA3 subfields of the hippocampus were countedseparately averaged over animals in a group, and expressed asmean number of cells/mm2 unit area. The protocol followed waspreviously described by Tandon et al. (1999).

2.7. Statistical analysis

Data were expressed as mean (N = 6) � S.E.M. Data analysis wasperformed by two-way ANOVA (using Graph Pad Prism software)with Bonferroni’s post hoc test for multiple comparisons. The level ofsignificance was accepted at p < 0.05. Pearson correlation wasperformed between different parameters and scatter plot matrix wasdrawn with the help of SYSSTAT software. Bonferroni test wasperformed to ascertain whether two parameters correlated sig-nificantly or not.

3. Results

The results suggest that long-term oral aluminium intakethrough drinking water results in alterations of hippocampalneuronal activity which closely correlates with the behavioral,biochemical and histological alterations.

3.1. Effect of Al-administration on MUA recordings

A two-way ANOVA (age � Al-toxicity) clearly indicate a sig-nificant age-related decrease of MUA activity [CA1 (F(1,20) = 312.47

Fig. 1. Electrophysiological recordings showing electroencephalogram (EEG), Integrated a

of hippocampus of control and aluminium-treated young and old rats.

Fig. 2. Effect of aging and oral aluminium intake related changes in multiple unit activity

Aging-associated decrease in the MUA activity and aluminium-induced increase in MUA a

(p < 0.05) in comparison to control young. p < 0.001***, p < 0.01**, p < 0.05* represent

p < 0.001); CA3 (F(1,20) = 45.22 p < 0.001)] whereas Al-toxicitysignificantly increased MUA activity [CA1 (F(1,20) = 146.96p < 0.001); CA3 (F(1,20) = 86.38 p < 0.001)]. A statistically significantinteraction was observed in MUA recordings of the CA3 (F(1,20) = 5.90p < 0.05), however, a non-significant interaction was observed inCA1 (F(1, 20) = 0.19 p > 0.05) field of hippocampus. Apart from MUAthe integrative amplitude (IA) of EEG was also significantly higher inAl-treated young and old rats compared to their respective agematched controls (Figs. 1 and 2).

3.2. Biochemical assays

3.2.1. Effect of Al on membrane lipids and membrane associated

proteins

Statistical analysis with two-way ANOVA (age � Al-toxicity)confirms that a significant interaction between age and Al-toxicityexists in lipid peroxidation (F(1,20) = 8.02 p < 0.01), Na–K ATPase(F(1,20) = 19.99 p < 0.001) and cytosolic PKC (F(1,20) = 11.50 p < 0.01)activity while a non-significant interaction was observed in boundPKC (F(1,20) = 1.14 p > 0.05) and AChE activity (F(1,20) = 0.45p > 0.05). In addition, analysis results clearly showed that long-term oral aluminium intake results in a significant increase in lipidperoxidation (F(1,20) = 68.20 p < 0.001), cytosolic PKC (F(1,20) =125.44 p < 0.001), bound PKC (F(1,20) = 188.73 p < 0.001) and AChE(F(1,20) = 53.96 p < 0.001), activity. Unlike other parameters Na–KATPase activity was significantly decreased (F(1,20) = 367.04p < 0.001) in aluminium-treated young and old rats compared totheir control (Fig. 3).

mplitude (IA) and multiple unit activity (MUA) from CA1 (A–D) and CA3 field (E–H)

recorded from CA1 (A) and CA3 (B) field of hippocampus of young and old rat brain.

ctivity is evident. Each data points represent mean SEM (n = 6). *Level of significance

s significant difference while $ represents non-significant difference.

Fig. 3. Effect of oral aluminium intake on lipid peroxidation (A), Na–K ATPase (B), protein kinase C (cytosolic) (C), protein kinase C (bound) (D) and acetylcholine esterase (E) in the

hippocampus of young and old rat brain. Aluminium-induced increase in LP, cytosolic PKC, AChE and decrease in Na–K ATPase activity and membrane bound PKC is evident. Each

value represents mean� S.E.M. (n = 6 animals from each group). p < 0.001***, p < 0.01**, p < 0.05* represents significant values while p > 0.05$ represents non-significant difference.

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–1079 1073

3.2.2. Effect of Al on antioxidative enzymes

A two-way ANOVA confirms that a significant interactionbetween age and Al-toxicity was observed only in SOD (F(1,20) =18.91 p < 0.001), while a non-significant interaction was observedin GPX (F(1,20) = 0.53 p > 0.05), and GST activity(F(1,20) = 0.25p > 0.05). We also found that aluminium intake significantlyinhibited the activity of antioxidative enzymes like SOD(F(1,20) = 61.04 p < 0.001), GST (F(1,20) = 48.80 p < 0.001) and GPx(F(1,20) = 83.63 p < 0.001). A significant age-linked decline in theantioxidative enzyme SOD (F(1,20) = 12.16 p < 0.01), GPx(F(1,20) = 21.72 p < 0.001), and GST (F(1,20) = 118.84 p < 0.001)activity was also observed (Fig. 4).

3.3. Behavioral tasks

3.3.1. Open field test

A two-way ANOVA and Bonferoni test confirms that asignificant interaction between age and Al-toxicity was observedin faecal index (F(1,20) = 16.34 p < 0.001) whereas non-significantinteraction exists in rearing (F(1,20) = 0.64 p > 0.05) and ambulatoryactivity (F(1,20) = 2.55 p > 0.05). Significant effect of age or Altoxicity was observed in both rearing (p < 0.01) and ambulatoryactivity (p < 0.01) is evident in Fig. 5.

3.3.2. Morris water maze test

Fig. 6a and b shows the average latency for both young and oldAl-treated groups to reach the fixed platform over each of 4 trial

days. A two-way ANOVA test confirms significant effect of Al-toxicity on latency to acquire hidden platform in both young(F(1,40) = 93.4 p < 0.001) and old (F(1,40) = 153.2 p < 0.001) group.Interaction between Al-toxicity and latency to acquire platform onconsecutive days of trials was found to be significant in both young(F(3,40) = 19.16 p < 0.001) and old (F(3,40) = 10.44 p < 0.001) groups.This confirms that oral Al-intake significantly retards the learningabilities of both young and old rats.

3.4. Histological observations

Statistical analysis confirms that Al-toxicity significantlydecreased number of cells in CA1 (F(1,12) = 35.43 p < 0.001) andCA3(F(1,12) = 470.41 p < 0.001) subfield of hippocampus. A two-way ANOVA and Bonferroni post hoc test confirmed a significantinteraction of Al-toxicity and age in CA3 (F(1,12) = 41.58 p < 0.001)field while non-significant interaction was seen in CA1(F(1,12) =0.65 p > 0.05) field. Qualitative studies of cresyl violet-stainedsections shows that pyramidal neurons of both aged and Al-toxicated group exhibited cellular shrinking, densely pigmentedcytoplasm and disorganized pyramidal cellular arrangements(Figs. 7 and 8).

3.5. Correlation between observed parameters

Aging-associated decrease of MUA activity positively correlatedwith the decrease of Na–K ATPase activity (r-value = +0.91), AChE

Fig. 4. Effect of oral aluminium intake on antioxidative enzymes: Superoxide

dismutase (A), glutathione-S-transferase (B), glutathione peroxidase (C) in the

hippocampus of young and old rat brain. Aluminium-induced decrease in SOD, GST

and GPx activity is evident. Each data point represents mean � S.E.M. (n = 6 animals

from each group). p < 0.001***, p < 0.01**, p < 0.05* represents significant values

while p > 0.05$ represents non-significant difference].

Fig. 5. Open field test results exhibit decreased rearing activity while increased

ambulatory activity and faecal index in Al-treated rats. Each point represents the

mean (n = 6) � S.E.M. p < 0.001***, p < 0.01**, p < 0.05* represents significant values

while p > 0.05$ represents non-significant difference.

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–10791074

(r-value = +0.80), cytosolic (r value = +0.74) and bound PKC,antioxidative enzymes (r-value = �0.86) and negatively correlatedwith lipid peroxidation (r-value = �0.93). Aluminium caused in anincrease of MUA activity, which negatively correlated withdecrease of antioxidative enzymes, Na–K ATPase (r-value = �0.93)0.93) activity while positively correlated with AChE (r-value = 0.8)and PKC (r-value = 0.75). Our correlative studies suggest thatdifferent parameters strongly correlated with each other (Figs. 9and 10).

4. Discussion

In the present study, we have investigated the effect of long-term intake of AlCl3 in hippocampus of young and old rats. Ourobservations indicate that aluminium potentially interferes withthe neuronal electrophysiological and behavioral outcome whichsignificantly correlated with the biochemical and histologicalalterations. In electrophysiological studies, multiple unit actionpotentials and integrated amplitude was extracted from the depthEEG recordings from CA1 and CA3 field. MUA represents anelectrophysiological marker of the activity of a population ofneurons and its alteration reflects the biochemical, physiological

and behavioral changes of neurons (Sharma et al., 1993; Singh andSharma, 2005). We observed that aluminium intake results inhyperfiring/hyperexcitablity in the depth EEG which was con-firmed by significantly increased multiple unit action potentialrecordings in both young and old Al-treated rats. The increasedMUA could be attributed to the altered function of GABA-Areceptor associated with aluminium toxicity (Trombley, 1998). AsGABA is an important inhibitory neurotransmitter, alterations inthe GABA-A receptor function lead to widespread changes ininhibitory circuits that may be responsible for the observedenhanced excitability (Figs. 1 and 2). At low concentrations,aluminium potentiates and at high concentrations inhibits GABA-mediated currents (Trombley, 1998). Similar, effects of zinc onGABA receptors was also reported in the literature (Bloomenthalet al., 1994; Trombley and Shepherd, 1994).

In behavioral tasks, open field test parameters indicate elevatedanxiety in the form of high ambulation and increased defecationindex in Al-intoxicated young and old rats. The defecation index isan indicator of potential anxiety (Suarez-Fernandez et al., 1999).Data obtained positively correlated with the increased MUAactivity and hyper-excitability recorded in the form of increasedintegrated amplitude. Thus, our electrophysiological observations

Fig. 6. Showing the escape latency of the young and old rats in Morris water maze

after chronic treatment of aluminium for 6 months. Water maze task was

performed to evaluate the spatial memory ability, for 4 days. Each data point

represents the mean (�S.E.M.) latency of the five trials for six rats performed in 1 day.

Rats (n = 6) treated with aluminium were unable to acquire the spatial learning

memory task as rapidly as controls in both young as well as old rats. *Level of

significance (p < 0.05) in comparison to control young. (*) Level of significance

(p < 0.05) in comparison to control old. p > 0.05$ N.S. w.r.t. control young; p > 0.05c

N.S. w.r.t. control old.

Table 1Percentage increase or decrease of different parameters in the hippocampus of

aluminium-treated rats

Parameters Young Al-treated

(%age change w.r.t.

young control)

Old Al-treated

(%age change w.r.t.

old control)

MUA (CA1) 38.41" 38.81"MUA (CA3) 22.06" 20.91"Lipid peroxidation 85.45" 41.00"Na–K ATPase 43.34# 26.93#Cytosolic PKC 33.29" 62.21"Bound PKC 19.35" 22.59#AChE 43.33" 35.84"SOD 52.96# 16.52#GPx 40.00# 34.10#GST 23.08# 26.88#Cell count (CA1) 44.57# 38.20#Cell count (CA3) 53.00# 36.59#

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–1079 1075

were further supported by behavioral open field tests. Morriswater maze tests were also performed to investigate the spatiallearning abilities of Al-treated rats. Our data suggests that Al-treated rats have significantly lower ability to memorize in spatiallearning tasks. These observations can also be linked with thealtered MUA recordings observed in CA1 and CA3 fields ofhippocampus. As both fields are responsible for memory con-solidation (Singh and Sharma, 2005), in fact, for short-termmemory formation, hippocampal CA3 neuronal network modifythe incoming information and transfer the modified message to theCA1 subfield via CA3–CA1 projections (Li et al., 1994). Several in

vitro studies on metal toxicity have consistently suggested thattrace metals like Pb2+, Zn2+ and especially aluminium (Al3+) canirreversibly inhibit voltage activated calcium channel currents(VACCCs) (Busselberg et al., 1993, 1994; Platt and Busselberg,1994a,b) and all glutamate ionotropic receptors (Platt et al., 1994).Possibly, reduction of voltage gated calcium channel currents atthe presynaptic site will reduce the transmitter release whilethe decline of the glutamate activated current will diminishthe induction of an excitatory postsynaptic potential at thepostsynaptic side. Therefore, alterations in synaptic transmissionin Al-treated group could be responsible for decreased synaptic

plasticity. These reports support our observed aluminium-inducedlower learning abilities of Al-treated young and old rats. Inaddition, aluminium-induced impairment of long-term potentia-tion and long-term depressions both in vitro and in vivo have beenreported by Platt et al. (1995). Cognitive deficit associated withaluminium administration can also be attributed to the glialreaction and pathological changes reported in the cholinergicfibres of rat brain (Platt et al., 2001). Aluminium induced alterationin hippocampal LTP is ambivalent, Platt et al. (1995) suggested that100 mM impairs hippocampal LTP while no effect on themagnitude or longetivitiy of LTP was observed by Gilbert andShafer (1996). The possible discrepancy in the results could havearisen from the differences in the experimental protocol. OurMorris-water maze test results clearly demonstrated declinedspatial learning abilities in aluminium-treated rats. The decrease inthe memory functions strongly correlated with increased MUAactivity and anxiety observed in open field tests. Therefore, alteredneuronal firing of CA1 and CA3 field may be one of the possiblereasons for the observed cognitive deficit.

Aluminium treatment confers pleiotropic effect on differentbiochemical parameters undertaken in this study. Our datashowed an increase of lipid peroxidation and a general decreasein cellular antioxidants and Na–K ATPase activity in accordancewith our previous reports (Jyoti et al., 2007; Kaur et al., 2003a,b).Our statistical analysis confirms that a significant interaction wasobserved in several parameters undertaken in this study. In fact,we observed a significant interaction that was mainly observed inthe CA3 field of hippocampus which shows that this region is moreaffected with aging and Al-neurotoxicity compared to the CA1field. In addition, the % age increase and decrease (Table 1) clearlyshows that magnitude of oxidative damage to lipids, membraneassociated proteins and antioxidative enzyme (SOD) activity weremore severe in young Al-treated group in comparison to old ones.This indicated that aluminium toxicity severely affects youngerage group than old ones. The observed discrepancy of lipidperoxidation in young Al-treated group must be attributed to theamount of peroxidizable lipids available for oxidative damage. Thegeneral decline in the antioxidative defense system must havebeen responsible for the increased lipid peroxidation observed inthe hippocampus of young and old rats. In addition, potentialabilities of Al-ions to bind and displace Fe3+/Fe2+ (Sakamoto et al.,2004) could result in iron-mediated enhanced lipid peroxidation.In this study we have also observed alteration in cytosolic andmembrane-bound form of PKC to analyze its two distinctpopulations (Fordyce and Wehner, 1993; Orr et al., 1992).Translocation of hippocampal PKC from cytosol to membranehas been reported after long-term potentiation (Akers et al., 1986)

Fig. 7. Light microscopic photographs showing cellular arrangement in CA1 and CA3 field of hippocampus, in different group of treated rats (magnification = 40�). Aluminium

and aging-associated neuronal degeneration is evident.

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–10791076

and similar translocation has been reported in the CA1 field ofhippocampus after classical conditioning in rabbit nictatingmembrane/membrane eyelid conditioning paradigm (Bankset al., 1988; reviewed by Wehner and Sleight, 1990). We observed

that aluminium toxicity dissociates the membrane bound PKC tocytosol. Increase of cytosolic PKC was at the expense of decrease ofbound PKC as these parameters are inversely correlated(r = �8.75). Cytosolic form of PKC is the inactive form (in vivo)

Fig. 8. Effect of aging and oral aluminium intake related changes on cell density in

the CA1 (A) and CA3 (B) field of hippocampus of young and old rat brain. Aging and

aluminium toxicity-associated decrease in the cell count is evident. Each data

points represent mean � S.E.M. (n = 6 animals from each group). P < 0.001***,

p < 0.01**, p < 0.05* represents significant values while p > 0.05$ represents non-

significant difference.

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–1079 1077

(Gopalakrishna and Jaken, 2000) and the observed increase ofcytosolic PKC activity signifies that dynamics of PKC translocationwas reversed by aluminium toxication. Therefore, reversal effect ofaluminium toxicity on PKC dynamics could be correlated with the

Fig. 9. Scatter plot matrix showing correlation between electrophysiological MUA (CA1 re

0.7–1.0 strongly correlated; 0.5–0.7 moderately correlated; 0–0.5 weakly correlated; (�

lower learning abilities observed in the Morris-water maze tasks.The observed PKC alteration also correlated with the increasedlipid peroxidation, which might be responsible for displacement ofbound PKC to cytosol in aluminium toxicity. Pearson correlationmatrix clearly indicates strong correlation between biochemicalparameters with behavioral and electrophysiological observations.

Among membrane-associated proteins, AChE activity wasobserved to be elevated in Al-intoxicated young and old rats.Our results were in accordance with the recent reports (Zatta et al.,1994, 2002). Controversial results are also available (Julka and Gill,1996; Platt et al., 2001) which might be attributed to the variety ofexperimental toxicological protocols (Zatta et al., 1994) or speciesspecific variation. The observed elevated activity of AChE could bedue to a direct effect of Al(III), as Zatta et al. (1994) proposed thatAl(III) can interact with the peripheral sites of AChE to modify thesecondary structure and eventually its activity. Unlike AChE, Na–KATPase activity was found to be declined in both young and old Al-intoxicated rats. As increased lipid peroxidation alters the lipidenvironment, it may also affect membrane’s Na–K ATPase activity.Na–K ATPase enzyme is involved in maintenance of ionic gradientsacross the membrane. Therefore, changes in its activity could beassociated with alterations in neuronal action potential firing(Riddle et al., 1993). In accordance to our study, AlCl3-induceddisruption of Na and K channel proteins has been reported in vitro

by Zhang et al. (2004).Our histological observations indicated that Al-treated groups

exhibited disorganized pyramidal cellular arrangement, densecytosolic staining and degenerating neurons in both CA1 and CA3fields. Histochemical results indicating dense cytosolic stainingcould be linked with the increased lipofuscin accumulation in Al-toxicity (Jyoti and Sharma, 2006) and aging (Brunk and Terman,2002). Quantitative analysis shows that Al-treated groups exhibit asignificant decrease of cell count in both CA1 and CA3 fields ofhippocampus. The observed cellular alterations in the CA1 and CA3fields are attributed to the increased lipid peroxidation anddecrease of antioxidative enzymes activity since these are stronglycorrelated. Oxidative inactivation of Na–K ATPase could be one of

gion) and biochemical parameters in aging (A) and aluminium-intoxicated rats (B):

) inversely correlated; (+) positively correlated.

Fig. 10. Scatter plot matrix showing correlation between electrophysiological MUA (CA1 region), Histochemical (cell count) and behavioral (open field parameters) in aging

(A) and aluminium-intoxicated rats (B): 0.7–1.0 strongly correlated; 0.5–0.7 moderately correlated; 0–0.5 weakly correlated; (�) inversely correlated; (+) positively

correlated.

P. Sethi et al. / NeuroToxicology 29 (2008) 1069–10791078

the reasons for enhanced neuronal excitability (Vasilets andSchwarz, 1993). Inhibition of Na–K ATPase has been previouslylinked with intracellular accumulation of Na+, which reverses thedirection of Na+/Ca2+ exchange and exacerbate intracellular Ca2+

accumulation (Dipolo and Beague, 1983; Akerman and Nicholls,1981) which could further increase lipid peroxidation, excitotoxi-city/apoptosis (Choi, 1993). As these hippocampal fields areresponsible for memory consolidation, we propose that the alteredcytomorphological alterations of pyramidal cells could be playingcrucial role in lower learning abilities of Al-treated rats.

In conclusion, our results show that aluminium intake impairsspatial learning abilities and increases anxiety by modifying brainfunctions at electrophysiological, biochemical and structurallevels. We have also observed the magnitude of aluminiuminflicted neurotoxicity was significantly higher in younger rats incomparison to older rats. Correlation studies indicate thataluminium’s neurotoxic effect modulate different aspects of brainfunctions including cognition and behavior.

Acknowledgement

Authors are thankful to the University Grant Commission forproviding necessary funding and fellowship for this project.

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