Introduction

Alteration in metal homeostasis in the brain has been perceived as one of the key factors in the progression of neurodegenerative diseases. Various metals like aluminium, lead, iron, copper and mercury have been observed to be involved in the pathogenesis of neurological disorders [1, 2]. But the world over, among various central nervous system disorders, Alzheimer’s has been adjudged as one of the most prevalent neurodegenerative diseases [3, 4]. Mounting evidences for the last few years have shown that Al is the most potential aetiological agent in causing Alzheimer’s disease [5, 6]. Even several microscopic, spectroscopic as well as biochemical investigations have clearly elucidated the correlation between Al and Alzheimer’s disease [7, 8]. Apparently, the exact mechanism through which Al manifests the progression of disease and the molecular basis of its interaction is rather sparse and remains to be fully explored.

Human exposure to Al is mainly attributed to its wide availability in nature and through manmade sources [9–11]. Al has been reported to mediate its neurotoxicity either through its direct or indirect interaction with the neurons [12–14]. Studies have revealed that high accumulation of Al in the brain can cause several neuropathological alterations, impairment in various neurotransmitter systems and progressively leads to behaviour deficits [15–17]. Scientists have also proven that Al exposure can disturb the normal functioning of antioxidant defences system, apoptotic machinery as well as carbohydrate and lipid metabolism in the brain [18–21].

Recently researchers have found that Al can disrupt the calcium homeostasis in neurons by interacting with calcium binding sites [22, 23]. Further, studies have also indicated that disturbance in calcium concentration in the brain is associated with the development of distinct patterns of neurodegeneration as well as responsible for memory and learning deficits [15, 24, 25]. Although several theories have been put forward to unravel the mechanism behind aluminium and calcium induced neurodegeneration but the exact mechanism is not clear till date.

On the other hand, Zn is an essential micronutrient in the body and is the second important transition metal in the brain [26, 27]. It plays an important role in keeping the integrity of the blood–brain barrier and maintaining the composition of membrane lipids as well as proteins of neurons [28, 29]. Studies have also indicated its neuromodulatory role in various signal transduction processes and ion channels [30, 31]. Further, decrease in its levels is also responsible for various neurological deficits and diseased conditions [32, 33]. Evidences have also shown that low zinc status in the brain can also affect the uptake of calcium by neurons, which can progress into neurological disorders [34, 35]. Zn has also been proven as an efficient agent against various toxic conditions induced by various xenobiotics and metals in the brain [36–38]. Many in vivo and in vitro studies have also indicated that Zn possesses antioxidant as well as anti-apoptotic activity in the brain [39, 40]. The present study was aimed to evaluate the role of Zn, if any, during aluminium-altered calcium homeostasis and signal transduction pathways in the rat brain. Further, we are the first group which has shown an association between zinc, calcium and aluminium during neurodegeneration in experimental model.

Materials and Methods

Chemicals

Aluminium chloride and zinc sulphate were procured from Merck Ltd., whereas 45Ca was procured from Board of Radiation Isotope Technology, Mumbai, (India). 3′, 5′-Cyclic adenosine monophosphate (cAMP) immunoassay kit and calpain activity assay kit were procured from Biovision (USA). Various antibodies were purchased from Merck (India), Genei (India) and Sigma (USA). All other chemicals were obtained from Sigma Chemicals Company (USA).

Experimental Design

Healthy male Sprague Dawley rats in the weight range of 140–160 g were obtained from the central animal house of Panjab University, Chandigarh, India. The animals were housed in polypropylene cages under a hygienic bed of husk in a well-ventilated animal room and were provided with standard animal feed obtained from Ashirwad Industries, Kharar, Punjab, India. The animals had a free access to diet and drinking water throughout the study. All the animals were kept at 25 °C under a standard regimen with a 12:12-h light–dark cycle (darkness 20:00–08:00 hours) and a relative humidity of 45 to 50 %. Before the beginning of various treatments, the rats were kept for acclimatization to experimental conditions for a period of 1 week. All the procedures were done in accordance with ethical guidelines for care and use of laboratory animals, which were approved by Institutional Animal Ethics Committee, Panjab University, Chandigarh, India.

To carry out the study, the animals of age 7–8 weeks were segregated into the following four groups and each group consisted of six animals: Animals of normal control group (untreated rats) were fed with normal diet and water ad libitum throughout the period of experimentation. Aluminium-treated animals were given aluminium chloride (AlCl3 was dissolved in drinking water) orally at a dose level of 100 mg/kg body weight [41, 42]. Zinc-treated animals were supplemented with zinc in the form of ZnSO4.7H2O in drinking water, at a dose level of 227 mg/l [43, 44]. Combined Al- and Zn-treated animals were given a combined treatment of Al as well as zinc in a similar manner, as was given to aluminium- and zinc-alone-treated group animals, respectively. All the treatments continued for a period of 8 weeks.

At the end of the treatment schedule, the animals were sacrificed by decapitation after anaesthetization by diethyl ether. The brains were immediately removed, placed in ice-cold isotonic saline and dissected into cerebrum and cerebellum.

Preparation of Synaptosomes

Synaptosomes were obtained from the cerebrum and cerebellum regions of the brain by discontinuous sucrose gradient centrifugation method of Gray and Whittaker [45]. Briefly, homogenates of cerebrum and cerebellum 10 % (w/v) were prepared in 0.32 M sucrose in 20 mM Tris–HCl (pH 7.4) and were centrifuged at 1,000×g for 10 min. The supernatants were further centrifuged at 12,500×g for 20 min to obtain crude mitochondrial pellets. The pellets were suspended in 0.32 M sucrose in Tris–HCl and layered over a gradient containing 0.8, 1.0 and 1.2 M sucrose. After centrifugation at 80,000×g for 2 h, the interfaces over 1.0 M sucrose containing synaptosomal fractions were removed. These were again pelleted by centrifugation at 80,000×g for 30 min and were re-suspended in physiological buffer, which constituted of synaptosomal preparation.

45Calcium Uptake in Synaptosomes

45Ca uptake was studied in polarized (low K+) and depolarized (high K+) synaptosomes by the method of Edelfors and Ravn-Jonsen [46]. Addition of depolarized (high potassium) medium results in opening of voltage-operated Ca2+ channels, which facilitates the entry of 45Ca into the synaptosomes. In this procedure, freshly prepared synaptosomal preparations (20 μl) were equilibrated at 25 °C for 10 min. Reaction was initiated by the addition of 100 μl of low K+ medium, i.e. un-stimulated (136 mM NaCl, 4.9 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 11 mM glucose and 20 mM Tris–HCl, pH 7.4) or high K+ medium, i.e. stimulated (38 mM NaCl, 106 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2, 11 mM glucose and 20 mM Tris–HCl, pH 7.4) to synaptosomes containing 45Ca, respectively. The samples were incubated for 5 min and were stopped by the addition of ice-cold lanthanum medium and later filtered through presoaked millipore filters (0.45 μm pore size, Sartorius, USA) under vacuum. The filters were washed thrice by adding lanthanum medium and dried, and the radioactivity absorbed onto the filters was counted using the scintillation cocktail on a liquid scintillation counter. The scintillator (1,000 ml) used was comprised of 60 g naphthalene, 4 g PPO, 0.2 g POPOP, 20 ml ethylene glycol and 100 ml methanol, and the volume was made 1,000 ml with 1,4 dioxane [47]. The uptake at zero time represented the nonspecific binding of 45Ca to the synaptosomes. The net potassium-induced 45Ca uptake (ΔK) was obtained by subtracting the un-stimulated 45Ca uptake from stimulated 45Ca uptake.

Preparation of Synaptic Plasma Membrane

Synaptic plasma membranes were prepared from cerebrum and cerebellum of the rat brain for the assay of membrane-bound enzyme, Ca2+Mg2+ ATPase by using a discontinuous sucrose density gradient-ultracentrifugation method of Jones and Matus [48]. Briefly, cerebrum and cerebellum were homogenized separately in nine volumes of 10 % (w/v) sucrose. The homogenates were centrifuged at 800×g for 20 min, and the supernatants so obtained were centrifuged at 10,000×g for another 20 min. The pellets were then washed with 10 % (w/v) sucrose to yield the crude mitochondrial fractions. The pellets were suspended in hypotonic buffer (5 mM Tris–HCl, pH 8.0) and were incubated at 0 °C for 30 min followed by homogenization. The lysates were made as 34 % (w/v) by the addition of appropriate amount of 48 % (w/v) sucrose solution. The upper phase of 28.5 % (w/v) sucrose was layered over the sample phase, and a small volume of 10 % (w/v) sucrose was overlaid onto this upper phase. The density gradients were centrifuged at 60,000×g for 110 min. The following fractions were recovered from each inter phase and the residual pellet. The upper white flocculent layer contained myelin, the middle gray band contained the synaptic plasma membrane and the brown pellet contained mitochondria.

Ca2+ ATPase

Ca2+ ATPase was assayed in synaptic plasma membranes according to the method of Desaiah et al. [49]. The Ca2+ Mg2+ ATPase was assayed in a reaction mixture containing Tris–HCl buffer, MgCl2, CaCl2, sample and ATP. ATPase activity was determined in the presence of ethylene glycol tetraacetic acid (EGTA), and this was subtracted from the total Ca2+Mg2+ ATPase activity in order to obtain the net Ca2+ ATPase activity. The reaction mixture was incubated at 37 °C for 15 min, and the reaction was stopped by the addition of trichloroacetic acid (TCA). The contents were centrifuged at 3,000×g for 10 min, and Pi released was estimated in the supernatant by following the method of Fiske and Subbarow [50].

Na+ K+ ATPase

The enzyme activity was measured by using the method of Wallach and Kamat [51]. In this method, three test tubes were employed per sample. First tube was for test, second was for control and the third was for ouabain-insensitive enzymes. To the first test tube of each sample, homogenate was added which was followed by buffer and then substrate (ATP) and finally double distilled water was added. To the second tube of each sample, double distilled water was added instead of sample, but in addition, TCA was added. To the third tube, sample was mixed with buffer, ouabain, substrate (ATP) and followed by addition of double distilled water. Tubes were then incubated for 15 min at 37 °C on water bath. Later TCA solution was then added to stop the reaction. The contents were further centrifuged at 3,000×g, and the inorganic phosphorous released was estimated by following the method of Fiske and Subbarow [50].

cAMP Levels

cAMP is an important “second messenger” and is a critical component of signal transduction pathways that are involved in many physiological processes. The levels of cAMP were estimated by using cAMP direct immunoassay kit (Biovision, USA). The assay is based on the direct competitive immunoassay for sensitive and quantitative determination of cAMP levels. Protein G-coated plates anchors polyclonal antibody to the plate. cAMP–horseradish peroxidase (HRP) conjugate directly competes with cAMP from the sample to bind to the cAMP-specific antibody on the plate. After incubation and washing, the amount of cAMP–HRP bound to the plate was determined by reading HRP activity at O.D. 450 nm. The intensity of O.D. 450 nm was inversely proportional to the concentration of cAMP in the samples.

$$ \mathrm{Absorbance}\ \left(450\kern0.5em \mathrm{nm}\right)\ \alpha\ 1/\mathrm{cAMP} $$

Calpain Assay

Calpain is a degrading enzyme whose activation depends upon the intracellular calcium levels. The calpain activity assay kit (Biovision, USA) specifically extracts cytosolic proteins without contaminations of cell membrane and lysosome proteases. Calpain enzyme was extracted by treating the brain samples with extraction buffer. Further, the extracted mixture was diluted with reaction buffer, and calpain substrate was added to the reaction mixture, which led to the breakdown of the substrate. The cleaved calpain substrate Ac-LLY-AFC (acetyl-Leu-Leu-Tyr-7-amino-4-trifluoromethylcoumarin) was determined fluorometrically. Ac-LLY-AFC emits blue light (λ max = 400 nm) upon cleavage of the substrate by calpain; free AFC emits a yellow-green fluorescence (λ max = 505 nm), which was quantified by using a fluorescence plate reader. Comparison of the fluorescence intensity from a treated sample with a normal control allows determination of the changes in calpain activity.

Intrasynaptosomal Free Ca2+ Levels

Intracellular free Ca2+ was determined in synaptosomes with the calcium sensitive indicator dye Fura-2/AM according to the method of Adamson et al. [52]. In this method, isolated synaptosomes (in physiological buffer) were loaded with 1 mM Fura-2/AM and were incubated at 37 °C for 1 h. The excess Fura-2/AM was removed by centrifugation. The fluorescence (F) at 340 to 380 nm excitation and 510 nm emission was measured, and [Ca2+] i was calculated according to the formula:

$$ {\left[{\mathrm{Ca}}^{2+}\right]}_i\kern0.5em =\kern0.5em \left[\left(F\kern0.5em -\kern0.5em {F}_{\min}\right)/\left({F}_{\max}\kern0.5em -\kern0.5em F\right)\right]\kern0.5em \times \kern0.5em {K}_{\mathrm{d}} $$

The K d for Fura free acid is 225 nm. Maximal fluorescence (F max) was measured after lysis of synaptosomes with SDS, and minimal fluorescence (F min) was measured in the presence of 5 mM EGTA.

Calcium Estimation

Calcium levels were estimated in both the cerebrum and cerebellum by using atomic absorption spectrometer (Perkin Elmer) following the wet acid tissue digestion method of Zumkley et al. [53]. Briefly, the brain samples were mixed with perchloric acid/nitric acid (1:4 in volume) mixture, followed by digestion in sand bath until a white ash or residue was formed. The residue so obtained was dissolved in 10 mM nitric acid and was used for elemental analyses.

Western Transfer Analysis

Western blotting analysis was done according to the method of Towbin et al. [54]. The protein expressions of stress marker genes were observed in the post-mitochondrial fractions using anti-phospholipase C (PLC) (1:500) (Merck, Bangalore), anti-inositol triphosphate (IP3) receptor (1:500) (Genei, Bangalore), anti-protein kinase A (PKA) (1:500) (Merck, Bangalore) and anti-protein kinase C (PKC) (1:250) (Sigma, Bangalore). Densitometric analyses of bands were done by using Image J software (NIH).

Light Microscopic Examination

To carry out the histological studies, small sections of cerebrum (cerebral cortex) and cerebellum from each of the normal control and treated animals were taken. Immediately after sacrificing the animals, perfusion was done with 4 % paraformaldehyde, and the brains were subsequently fixed in 10 % formalin. After fixation, the tissues were processed carefully for embedding with paraffin wax (58–60 °C). Five- to seven-micrometre-thick paraffin sections were cut and were then subjected to alizarin red staining. Further, the stained slides were analysed under light microscope at ×10 and ×40 magnifications for histological examination.

Alizarin Red Staining

Alizarin Red S is an anthraquinone derivative used to identify calcium in tissue sections. In this staining, calcium forms an Alizarin Red S-calcium complex through a chelation process, and the end product formed is birefringent. In this staining procedure, the brain sections were de-paraffinized and brought to distilled water. Slides were stained with alizarin red solution (2 min) and were observed under microscopic (red-orange-stained calcium deposits). Excess of dye was removed by blotting, and slides were further dehydrated in acetone (five dips) followed by acetone-xylene (1:1) solution (five dips) and finally mounted with DPX.

Protein Estimation

Protein contents were estimated by using the method of Lowry et al. [55].

Statistical Analysis

The statistical significance of the data was determined by using one-way analysis of variance and a multiple post hoc test (Student–Newman–Keuls). The results were represented as mean ± SD of six observations. Comparisons were made as follows:

  • *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by Newman–Keuls test when values of treated groups are compared with normal control group

  • ****p ≤ 0.05, *****p ≤ 0.01, ******p ≤ 0.001 by Newman–Keuls test when values of Al + Zn group are compared with Al-treated group

Results

Synaptosomal K+ Stimulated 45Ca2+ Uptake

Effect of Al exposure on Ca2+ influx through voltage-operated calcium channels under stimulated (high K+) and unstimulated (low K+) conditions was analysed during our study. Two months of Al exposure to rats resulted in a significant increase (p ≤ 0.001) in Ca2+ influx in comparison to untreated control rats, which was observed via voltage-operated calcium channels, after incubation with radiolabelled calcium in both polarizing and depolarizing media (Table 1). Zn supplementation to Al-treated rats significantly reduced the calcium influx when compared to Al-treated rats and hence afforded protection in maintaining the low intracellular calcium levels. However, Zn treatment alone to normal rats did not reveal any statistically significant change in the calcium influx when compared to untreated control rats.

Table 1 Effect of zinc on net 45Ca uptake in synaptosomes isolated from cerebrum and cerebellum of rats subjected to aluminium treatment
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Ca2+ ATPase

Ca2+ATPase, a CaM-regulated membrane bound enzyme, is responsible for maintaining intracellular calcium levels in neurons. A significant decrease in the Ca2+ ATPase activity was observed following Al treatment in comparison to untreated control rats (Table 2). Interestingly, Zn co-treatment to Al-exposed rats caused a significant increase in the Ca2+ ATPase activity in cerebrum (p ≤ 0.010) and cerebellum (p ≤ 0.050) when compared to Al-treated rats. Further, no significant change was observed in the Ca2+ATPase activity when rats were treated with Zn in comparison to normal control rats.

Table 2 Effect of zinc on Ca2+ ATPase activity in cerebrum and cerebellum of rats subjected to aluminium treatment
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Na+/K+ ATPase

Al treatment significantly inhibited Na+/K+ ATPase enzyme activity in both cerebrum and cerebellum (p ≤ 0.001) in comparison to untreated control rats (Table 3). However, when Al-treated rats were supplemented with Zn, a significant increase in the Na+/K+ ATPase activity was observed in cerebrum (p ≤ 0.050) and cerebellum (p ≤ 0.010) as compared to Al-treated rats. Further, zinc alone treatment did not cause any significant change in the Na + /K + ATPase activity in both regions of the brain when compared to untreated control rats.

Table 3 Effect of zinc on Na+/K+ ATPase enzyme activity in cerebrum and cerebellum of rats subjected to aluminium treatment
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cAMP Levels

The levels of cAMP were found to be elevated in synaptosomes of cerebrum (p ≤ 0.010) and cerebellum (p ≤ 0.001) of Al-exposed animals in comparison to untreated normal control rats (Table 4). On the contrary, Zn supplementation to Al-treated animals significantly declined the cAMP levels in synaptosomes of both the cerebrum (p ≤ 0.050) and cerebellum (p ≤ 0.001) when compared to Al-treated rats. However, no significant change in the levels of cAMP was observed in rats treated with zinc alone as compared to control rats.

Table 4 Effect of zinc on cAMP levels in synaptosomes isolated from cerebrum and cerebellum of rats subjected to aluminium treatment
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Calpain Activity

Calpain, a degrading calcium-dependent enzyme, is a cysteine protease, and its activity is directly related to the intracellular calcium levels. After 2 months of Al exposure, a significant increase (p ≤ 0.001) in calpain activity was observed in both the cerebrum and cerebellum when compared to normal control rats (Table 5), whereas simultaneous Zn treatment to Al-treated rats significantly decreased the calpain activity in both regions of the brain in comparison to Al-exposed rats.

Table 5 Effect of zinc on calpain activity in cerebrum and cerebellum of rats subjected to aluminium treatment
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Intrasynaptosomal Free Ca2+ Levels

Al exposure resulted in an increased (p ≤ 0.001) intracellular free calcium levels in synaptosomes of cerebrum and cerebellum when compared to free intracellular calcium levels of untreated control rats (Table 6). However, zinc co-administration was able to reverse the trend in synaptosomes of both regions of the brain, and the levels were appreciably reduced in comparison to Al-treated rats. Further, zinc alone treatment did not cause any statistically significant change in intracellular free calcium levels in synaptosomes of cerebrum and cerebellum when compared with normal control rats.

Table 6 Effect of zinc on intrasynaptosomal free [Ca2+] i in cerebrum and cerebellum of rats subjected to aluminium treatment
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Calcium Levels

A significant increase in the levels of Ca was observed in cerebrum (p ≤ 0.010) and cerebellum (p ≤ 0.001) after 2 months of Al exposure when compared to the untreated normal control rats (Table 7). Zinc supplementation to Al-treated rats significantly reduced the levels of Ca in cerebrum (p ≤ 0.050) and cerebellum (p ≤ 0.010) in comparison to rats treated with Al alone. However, no significant change in the Ca levels was observed in Zn-treated animals when compared to untreated control rats.

Table 7 Effect of zinc treatment on the levels of calcium in cerebrum and cerebellum of rats subjected to aluminium treatment
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Western Transfer Analysis

Two months of Al treatment resulted in a significant increase (p ≤ 0.001) in the protein expressions of PLC, IP3 and PKA whereas decreased expression of PKC in both the cerebrum and cerebellum when compared with untreated control rats (Fig. 1). On the contrary, Zn co-administration to Al-treated rats resulted in a significant decrease in the protein expressions of PLC, IP3 and PKA and increased expression of PKC in comparison to Al-alone-treated rats. Further, Zn alone treatment caused no significant change in the protein expressions of PLC, IP3, PKA and PKC when compared to untreated normal control rats.

Fig. 1
figure 1

Western blot and densitometric analyses of phospholipase C (PLC), inositol triphosphate (IP3), protein kinase A (PKA), protein kinase C (PKC) and β-actin in the cytosol isolated from cerebrum and cerebellum of normal control, aluminium-, zinc- and aluminium + zinc-treated animals

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Light Microscopic Observations

Figures 2a and 3a show normal control sections of cerebrum and cerebellum. Calcium staining with alizarin red S in Al-treated brain sections showed increased red-orange-coloured deposits of calcium in both regions of the brain (Table 8). Elevated alizarin red-positive staining revealed the high content of calcium in the brain tissue of Al-treated animals, which suggests the neurodegenerative conditions in the brain due to calcium dyshomeostasis (Fig. 2b and 3b). Zinc-treated brain sections of cerebrum and cerebellum had similar morphology, as that for normal control sections of the brain (Fig. 2c and 3c). Further, combined Al and Zn rat brain sections showed lesser deposits of calcium in comparison to Al-treated brain sections (Fig. 2d and 3d).

Fig. 2
figure 2

The calcium localisation in cerebrum section of rat from different treatment groups. a Normal histoarchitecture of cerebrum with no calcium deposits in normal control group. b The section represents the deposition of calcium (marked as black arrows) after aluminium treatment whereas c a cerebrum section from zinc-treated group with no calcium burden. Cerebrum section from combined treatment group d shows improvement with reduced calcium deposits

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Fig. 3
figure 3

a Section from normal control rat represents normal morphology of cerebellum with no calcium burden; b cerebellum from aluminium-intoxicated rat with red-coloured alizarin-positive deposits of calcium (black arrows); c section of cerebellum from zinc-treated group with normal histoarchitecture of the brain similar to that of control d rat cerebellum section from Al + Zn-treated group with lesser deposits of calcium in comparison to Al-alone-treated brain section

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Table 8 Number of calcium deposits in the brain of rats subjected to different treatments
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Discussion

Accumulated evidences have shown that altered calcium ion concentration in the brain can affect the functioning of numerous metabolic processes of central nervous system [25, 56, 57]. Calcium plays an important role in regulating various neuronal processes, including excitability, neurotransmitter release, gene transcription, cell proliferation and synaptic plasticity [58, 59]. Further, maintaining normal calcium content in a cell either through influx or efflux mechanism has been considered as a vital phenomenon for normal neuronal activity. On the other hand, aluminium after crossing the BBB is known to antagonistically replace various essential elements such as calcium, magnesium, zinc and phosphorus from various enzymes and proteins that are present in the brain [1, 9, 60]. This impairment in elemental status induced by Al has been reported to cause adverse effect in the brain’s neurophysiology by altering a series of biochemical cascades [15, 16, 21]. The present study has been designed to assess the effect of Al on calcium signalling pathway and also to evaluate the possible protection, if any, provided by zinc in such conditions.

Ca2+ ATPase is a membrane-bound transport protein that helps in regulating intracellular calcium levels by removing excess of Ca2+ from neuronal cells. In the present study, the activity of Ca2+ ATPase was found to be significantly decreased after Al exposure that reflects the impairment in Ca2+ extruding capacity of the cell and is understandably due to direct interaction of Al on the active site of Ca2+ ATPase [61]. It has been reported that Ca2+ ATPase requires Mg2+ATP complex as a cofactor to attain its normal activity [62]. Al due to its high affinity for Mg2+ (possess same physiochemical characteristics) would have replaced it from Mg2+ ATP complex and formed Al-ATP complex, which is further a potent inhibitor of Ca2+ ATPase [63]. Studies have also suggested that Al can decrease the activity of calcium ATPase by altering the conformation/activity of calcium binding protein calmodulin [22, 64, 65]. However, Zn treatment was significantly able to improve the activity of calcium ATPase in both regions of the brain (Table 2). This is because Zn might have prevented the attachment of Al to the active binding site of Ca2+ ATPase/Mg2+ATP or its supplementation has regulated the disturbed activity of calmodulin. Law et al. [66] have also shown a significant role of zinc in regulating calmodulin activity during zinc-deficient conditions.

Further, our results showed a significant increase in intrasynaptosomal free calcium level in cerebrum and cerebellum after aluminium treatment. This substantial increase is apparently due to decreased Ca2+ exuding ability as a result of decreased calcium ATPase activity. The increase in calcium levels may also be due to release of calcium from intracellular stores, a process reported to be triggered in the presence of metals [67]. Further, increase in total calcium levels has also been observed in our study after Al treatment, which suggests the overall disturbance in calcium content of neurons (Table 7). Further, increased calcium ions stress in neurons can also trigger the process of lipid peroxidation, which also justifies our findings as increased LPO levels have also been seen in our study after Al exposure (data not shown). However, Zn treatment to Al-exposed animals was able to decrease the elevated calcium levels in the brain, which is due to scavenging activity of Zn. Hence, Zn by quenching the Al-induced free radicals as well as free calcium ions from neurons has resulted in the reduction of cellular stress and accelerated a number of calcium- as well as non-calcium-dependent cellular mechanisms [29, 39, 68]. Further, its role in maintaining metal ionic concentration has also been studied in various neurotoxic conditions, which supports our findings [69, 70].

Reports have also suggested that raised intracellular calcium concentration in the brain can also be due to default in influx mechanism [57, 59]. So, we have also studied the effect of Al treatment on 45Ca2+ influx through voltage-operated calcium channels under stimulated (high K+) and unstimulated (low K+) conditions. Aluminium exposure for 2 months resulted in a significant increase in 45Ca influx in both the cerebrum and cerebellum. These findings suggest the harmful effect of Al3+ on voltage-operated calcium channels, which plays a crucial role in coupling electrical activity during the process of neurotransmission and open transiently in response to membrane depolarization. This enhancement could further lead to the raised levels of intracellular calcium, thus causing calcium stress in neurons and thereby leading to altered calcium homeostasis [61]. Another possible reason for this stress is due to Al3+-induced perturbations in the structure of the channel molecule or in a phospholipid domain, which has potential to alter the calcium transport capacity and causing oxidative injury in neurons [71]. Recently, Walton [72] proposed that Al by inhibiting the activity of Ser/Thr phosphatase impedes Ca2+ influx mechanism in NMDA receptors. Further, Zn co-treatment with Al proved beneficial as it was able to reduce the calcium influx in both regions of the brain. It seems reasonable that Zn understandably has protected protein thiol groups of calcium channel from getting oxidized as increased calcium ion concentration in neurons might have caused oxidation of calcium channels [73]. Further, Zn has been considered as a potent antioxidant during various adverse physiological conditions and therefore could have afforded protection against calcium-induced neuronal injury by regulating the normal functioning of calcium channels [28, 36, 39, 43]. Moreover, our previous findings have also shown that Zn is a potential agent for alleviating Al-induced oxidative DNA injury [41]. Other probable alternative is that Zn by competing with calcium for its binding site within the channel or close to its vicinity has inhibited the calcium influx and accelerated the process of synaptic transmission, which is also observed in our study (data not shown). Further, Kitamura et al. [74] and Franco-Vidal et al. [75] have also shown that Zn helps in reducing calcium influx mechanism during ischemic neuronal injury and pneumolysin-induced toxicity in rats.

Further, to assess the effect of Al on intracellular signal transduction pathways, cAMP levels were also determined in the present study. cAMP, a second messenger, plays an important role in many biological processes such as regulating the functions of ion channels. In the present study, after 2 months of Al exposure, a significant increase in the levels of cAMP was found in both regions of the brain which is apparently due to indirect effect of raised intracellular Ca levels (Tables 4 and 6). The plausible explanation for this is that increased intracellular calcium levels in neurons after Al treatment could have activated Ca/calmodulin-dependent adenyl cyclase activity, which further triggered the cAMP [76]. Few researchers have also observed disturbed cAMP activity after aluminium exposure in rats and in other in vivo studies [65, 76]. However, Zn co-treatment was able to decrease the raised calcium levels in both regions of the brain. The decrease in calcium levels due to zinc supplementation might have led to deactivation of calcium/calmodulin dependent adenyl cyclase, thereby reducing the levels of cAMP. Similar inhibitory effect of Zn on cAMP levels has also been reported by Klein et al. [77].

Calpain is a calcium-dependent degradative enzyme, which belongs to cysteine protease family and gets activated during adverse conditions such as mitochondrial dysfunctioning and disruption in calcium homeostasis. Our data indicate that after 2 months of aluminium exposure, there was a significant increase in the enzyme activity in both the cerebrum and cerebellum, which is attributed to increased level of calcium in neurons (Table 5). It may be argued that enhanced activity of this degradative enzyme leads to neuronal degeneration as a consequence of Al exposure. Similar effect on calpain activity has also been observed by Kaur and Gill [61] in different regions of the brain after intragastrical administration of Al in rats. However, Zn supplementation proved to be effective as it was able to reduce the calpain activity, thereby indicating maintenance of calcium homeostasis by zinc. Similar inhibitory activity of Zn on calpain activity has been reported by Whipple and Koohmaraie [78] during in vitro studies. In addition, increased calpain activity in the present study is an indicator of Al-induced neuronal apoptosis, which, however, was contained appreciably by Zn (data not shown). Zn also exhibits its anti-apoptotic property and thus could have afforded protection by acting at calpain, thereby stabilising the mitochondrial integrity as well as the resistance of neurons towards free radical-induced apoptosis [27, 40].

Phospholipase C pathway is one of the signalling pathways, which can cause increase in calcium content in the cells. PLC is a class of enzymes that cleaves phospholipids. It hydrolyses the membrane phospholipid PIP2 and cleaves into two second messengers via inositol 1,4,5-trisphosphate and diacylglycerol (DAG). IP3 is soluble in nature due to which it can diffuse into the cytosol and binds to its receptor where it stimulates the release of calcium ions. On the other hand, DAG remains in the plasma membrane and is a physiological activator of PKC. During our study, Al treatment increased the activity of PLC, IP3 and PKA whereas it down-regulated the activity of PKC in both regions of the brain (Fig. 1). The probable mechanism behind this is that Al induced membrane depolarization that might have activated the G-protein cascade and led to activation of catalytic subunit of PLC (PLCγ1), which could have resulted in the breakdown of PIP2. Further, G-proteins are involved in the regulation of ion channels, metabolism and cytoskeletal structures that were also found to be altered in the present study. Increased activity of IP3 is further responsible for release of calcium into the cell from intracellular stores (ER and mitochondria), which ultimately causes increased calcium concentration in the cell. Further, IP3 can also activate Ca2+ channels on the cell membrane indirectly by increasing the intracellular Ca2+ concentration as observed in our study following Al exposure [79]. Several studies have also shown the adverse effects of Al on G-protein signalling pathways that further caused calcium dyshomeostasis [72, 80]. Further, increased activity of protein kinase A has also been observed in the present study after Al treatment. PKA belongs to a family of enzymes whose activity depends on cellular levels of cAMP. Furthermore, increased levels of cAMP have also been noticed in our study, which clearly indicates the reason for raised activity of PKA [81]. Zundorf and Reiser [56] have also suggested similar findings during various neurodegenerative conditions. During our study, Al treatment caused a decline in PKC activity, which is a consequence of direct inhibitory action of Al on active sites of PKC. Al apparently has attacked on any of the three binding sites (Mg2+-ATP, DAG and calcium) of PKC and has led to a decrease in the PKC activity by successfully competing with Mg2+ for ATP and also by blocking Ca2+ binding to the carboxyl group on PKC [82]. Another possible reason for decreased activity is due to reduced activity of DAG, which further has caused declined activity of PKC. Further, similar effect of Al exposure on PKC activity has been shown by Cochran et al. [83] and Katsuyama et al. [84]. Therefore, from these findings, it can be inferred that Al treatment creates an imbalance in the calcium ion homeostasis, by affecting the voltage-activated calcium release, Ca2+ATPase activity and PLC activation, thus disrupts the second messenger pathways.

Zinc supplementation, however, regulated the disturbed calcium homeostasis and G signalling cascade in the neurons. Decreased PLC and IP3 activity after Zn treatment shows its regulatory effect on G protein signalling, which is reasonably due to reduction of Al-induced calcium load and cellular stress that is very apparent from our previous findings. Similarly, Jansen et al. [85] have also shown the effectiveness of Zn in regulating PLC activity in C6 glioma cells, whereas declined PKA activity is directly correlated to the decreased cAMP levels after Zn treatment. The possible reason for improvement in activity of PKC is due to displacement of Al from active binding sites of PKC by zinc. Moreover, protein kinase C is a Zn metalloenzyme, and its supplementation might have also up-regulated its declined activity.

Further, decrease in Na+/K+-ATPase activity has also been observed in both the cerebrum and cerebellum after Al treatment, which reflects an imbalance in ion homeostasis of neurons. The decline in this transmembrane protein activity is possibly due to direct attachment of Al with the subunits of Na+/K+ ATPase, or Al-induced free radicals have caused energy-depleted conditions in a cell [20, 23]. Failure of the Na+/K+ ATPase has been implicated in the pathophysiology of neurodegenerative diseases. Various studies have also described the critical role of Na+/K+ ATPase in signal transduction and cell death pathways (apoptosis, necrosis and hybrid cell death) [86, 87]. However, Zn treatment to Al-treated rats significantly increased the Na+/K+ ATPase enzyme activity (Table 3). This might be the result of displacement of Al by zinc from the active binding site of enzyme, or its strong antioxidant behaviour has regulated ionic metabolism in neurons. Also, Lovell et al. [88] have shown the protective effect of Zn against Abeta neurotoxicity by increasing the activity of Na+/K+ ATPase that helps in maintaining the calcium homeostasis and prevents cell death.

Histopathological studies have also revealed the increased content of calcium in the brain after Al treatment for 2 months (Fig. 2, Fig. 3 and Table 8). A number of alizarin-stained red-coloured calcium deposits were seen after Al treatment, which complements the observed increase in intracellular calcium content, activated proteases and lipases in the present study. On the other hand, stained sections from Zn co-treated group have shown reduced number of calcium deposits, thereby suggesting the regulatory role of zinc on calcium content. Further, improvement in calcium levels and activities of various calcium regulating proteins also suggests the positive influence of Zn on calcium homeostasis.

In conclusion, the findings of the current study suggest that zinc is a potential neuromodulatory agent and effectively maintains calcium homeostasis during Al-induced neurodegeneration.