Nicotine attenuates spatial learning deficits induced by sodium metavanadate

Nicotine attenuates spatial learning deficits induced by sodium metavanadate

NeuroToxicology 33 (2012) 44–52 Contents lists available at SciVerse ScienceDirect NeuroToxicology Nicotine attenuates spatial learning deficits ind...

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NeuroToxicology 33 (2012) 44–52

Contents lists available at SciVerse ScienceDirect

NeuroToxicology

Nicotine attenuates spatial learning deficits induced by sodium metavanadate Kian Azami a, Kaveh Tabrizian b, Rohollah Hosseini c, Mohammad Seyedabadi a, Marjan Shariatpanahi a, Farshid Noorbakhsh d, Abbas Kebriaeezadeh a, Seyed Nasser Ostad a, Mohammad Sharifzadeh a,e,* a

Department of Pharmacology and Toxicology, Pharmaceutical Sciences Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran Faculty of Pharmacy, Zabol University of Medical Sciences, Iran c Basic and Clinical Toxicology Research Center, Tehran University of Medical Sciences, Tehran, Iran d Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran e Department of Neuroscience, Faculty of Advanced Science and Technology in Medicine, Tehran University of Medical Sciences, Tehran, Iran b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 July 2011 Accepted 10 November 2011 Available online 19 November 2011

Learning can be severely impaired as a consequence of exposure to environmental pollutants. Vanadium (V), a metalloid which is widely distributed in the environment, has been shown to exert toxic effects on a variety of biological systems including the nervous system. However, studies exploring the impact of vanadium on learning are limited. Herein, we investigated the effects of oral administration of sodium metavanadate (SMV) (15, 20 and 25 mg/kg/day for 2 weeks) on spatial learning using Morris water maze (MWM). Our results showed that pre-training administration of sodium metavanadate impaired learning in Morris water maze. Analyzing the role of cholinergic system in SMV-induced learning deficit, we found that bilateral intra-hippocampal infusion of nicotine (1 mg/side) during training could significantly diminish the SMV-induced learning impairment. We next examined the expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) as cholinergic markers in CA1 region of hippocampus as well as in medial septal area (MSA). Our molecular analyses showed that vanadium administration decreased ChAT and VAChT protein expression, an effect that was attenuated by nicotine. Altogether, our results confirmed the toxic effects of SMV on spatial acquisition, while also pointing to the neuroprotective effects of nicotine on SMV-induced impairments in learning capabilities. These findings might open a new avenue for the prevention of vanadium adverse effects on spatial learning and memory through activation of cholinergic signaling pathway. ß 2011 Elsevier Inc. All rights reserved.

Keywords: Sodium metavanadate Nicotine Choline acetyltransferase (ChAT) Vesicular acetylcholine transporter (VAChT) Hippocampus Spatial learning

1. Introduction Vanadium (V) is a transition metal, which is widely distributed in the environment. The increase of vanadium levels in atmosphere is a consequence of various industrial activities including combustion of fossil fuels such as gasoline and coil (CalderonGarciduenas et al., 2002; Fortoul et al., 2002). Indeed, vanadium is considered as the most abundant metallic ingredient in petroleum (Amorim et al., 2007). Hence, about 75% of 265,000 tonnes of annual discharge of vanadium to the environment originate from human activities (WHO, 2001). This metal is used as a catalyst in

Abbreviations: SMV, sodium metavanadate; MWM, morris water maze; ChAT, choline acetyltransferase; VAChT, vesicular acetylcholine transporter; nAChRs, nicotinic acetylcholine receptors; MSA, medial septal area; MAPK, mitogenactivated protein kinase; CREB, cAMP response element-binding; PKA, protein kinase A; AChE, acetyl cholinesterase. * Corresponding author at: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran. Tel.: +98 21 66482705; fax: +98 21 66482705. E-mail address: [email protected] (M. Sharifzadeh). 0161-813X/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2011.11.004

many industrial processes including the production of plastic and sulfuric acid (Friberg, 1986). It is also used in production of steel, temperature resistant alloy products, glass, pigment, paint and super-conductive materials (Kaul et al., 2003; McNeilly et al., 2004; Robert, 2006). This leads to enhanced exposure to vanadium in certain occupations including power plant makers, boiler makers and oil refinery workers (Fortoul et al., 2002; Levy et al., 1984; Woodin et al., 1999, 1998, 2000). Vanadium compounds have been demonstrated to exert antihypertensive (Bhanot and McNeill, 1994), anti-diabetic (Bhanot and McNeill, 1994; Cam et al., 1995; Cohen et al., 1995; Heyliger et al., 1985), anti-hyperlipidemic (Pugazhenthi et al., 1995), and anticancer effects (Cruz et al., 1995; Kanna et al., 2003). However, toxic properties of these compounds are also well established. For instance, vanadium exposure in mammals has been reported to cause weight loss (Sanchez et al., 1998), morphological and biochemical changes in different organs (al-Bayati et al., 1989), thrombocytopenia (Gonzalez-Villalva et al., 2006), male reproductive system toxicity (Fortoul et al., 2007), hematological abnormalities (Zaporowska and Wasilewski, 1989), and neurotoxicity in the central nervous system (CNS) (Garcia et al., 2005). Despite the

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widespread applications of vanadium, the impact of this metal on health, and in particular its CNS effects are not well characterized. Zhou et al. (2007) have shown that exposure to vanadium can lead to decreased visual memory, mood disorder and motor disturbance in workers (Zhou et al., 2007). While vanadium toxicity in human chiefly manifests as CNS depression and tremor (Afeseh Ngwa et al., 2009), in animal models, it impairs learning and spatial memory (Avila-Costa et al., 2006; Mao et al., 2008; Sanchez et al., 1998). There is also some evidence that indicated hippocampal CA1 damage was induced by vanadium exposure in male mice (Avila-Costa et al., 2006). Hippocampus, part of the limbic system, exerts a key role in learning as well as memory formation in rats and other mammals (Abel et al., 1997; Baulieu and Robel, 1990). In humans, hippocampal damage leads to deficits in learning about locations, people, and objects (Vitolo et al., 2002). Indeed, spatial memory predominantly depends on this structure (Avila-Costa et al., 2006; Broadbent et al., 2004; Markowska et al., 1993; Olton and Papas, 1979). In rodents a convenient instrument for examining the spatial learning and memory is Morris water maze (MWM) (D‘Hooge and De Deyn, 2001). It is widely believed that hippocampal cholinergic neurons and in particular, the nicotinic acetylcholine receptors (nAChRs) expressed by these cells, play a crucial role in memory formation and cognition (Decker et al., 1995; Hiramatsu et al., 2002; Kim and Levin, 1996; Puma et al., 1999; Stolerman et al., 1995). Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT), which are involved in the synthesis, storage and release of acetylcholine (Ach), are expressed in specific neuronal systems (Berse and Blusztajn, 1995). Nicotine, by enhancing the ACh release (Hiramatsu et al., 2002; Stolerman et al., 1995) improves memory in mice and rats (Haroutunian et al., 1985; Sansone et al., 1991; Zarrindast et al., 1996). There are also reports pointing to the protective effect of nicotine on memory deficits caused by nicotine antagonists (Zarrindast et al., 1996) or brain lesions (Decker et al., 1992; Grigoryan et al., 1994). It has also been reported that nicotine can improve memory by enhancing hippocampal synaptic transmission (Hiramatsu et al., 1994, 2002; Whitehouse and Kalaria, 1995). Since there is little information about the hazardous concentrations as well as the mechanism(s) by which vanadium exposure leads to spatial learning deficits, in this study we investigated the effects of different doses of oral sodium metavanadate (SMV), together with its interaction with intrahippocampal infusion of nicotine, on spatial acquisition using the MWM method. Expression of ChAT and VAChT proteins in the CA1 region of hippocampus as well as in medial septal area (MSA) were also evaluated to show the possible relation between cholinergic markers and behavioral results. 2. Materials and methods 2.1. Drugs Sodium metavanadate and nicotine hemisulfate were purchased from Sigma (St. Louis, Mo, USA). Ketamine and xylazine, required for the induction of anesthesia, were obtained from Alfasan (Utrecht, Holland). 2.2. Animals Male Albino-Wistar rats (200–220 g) were purchased from Pasteur Institute of Iran (Tehran, Iran). The animals were housed in groups of four in cages (41 cm long, 28 cm wide, 15 cm high) and were maintained at room temperature (25  2 8C) under a 12 h light– 12 h dark cycle. All steps of animal training and testing were performed during the light cycle. Normal rat chow and water were provided ad

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libitum and all animal experiments were carried out in accordance with the guidelines of the Ethical Committee for the Care and Use of Laboratory Animals of Tehran University (357; 8 November 2000). 2.3. Behavioral training In all experimental groups, the animals were trained in the Morris Water Maze. The maze included a black-painted circular pool (136 cm diameter, 60 cm height), filled to a depth of 35 cm with water (25  2 8C). The pool was divided into four quadrants with four starting points, i.e. north (N), south (S), east (E) and west (W), all at equal distance from the edge. An invisible platform (10 cm in diameter), made of Plexiglas was immersed 1 cm under the water surface at the center of North-West quadrant (target quadrant). The rats were trained for 4 days. Each training session included one block, which was consisted of four trials. Each trial was started by placing the animal randomly in one of the four starting points. Animals were allowed to swim in pool during a period of 90 s to find the hidden platform. If an animal did not find the hidden platform within this period, it was manually guided to platform by the researcher. The rats then rested for 30 s on the platform between two consecutive trials. All trials were performed at about the same time of the morning. Swimming trajectories were recorded by a video camera linked to a computer. Spatial acquisition was evaluated by measuring escape latency (time to find the platform), traveled distance (path length to reach the platform), and swimming speed using the EthoVision tracking system (Noldus Information Technology, Wageningen, The Netherlands), as described in our previous works (Sharifzadeh et al., 2005a,b,c, 2007; Tabrizian et al., 2010). Sensorimotor coordination and motivation toward a visible platform was evaluated by visible platform task, including 1 block of 4 trials. Performance on the visible platform was similar to other trials. The difference was that the platform was covered with a piece of aluminum foil and elevated above water level in the center of opposite quadrant of the previously hidden platform. The visible platform manipulation was introduced one day after completion of training (day 5). 2.4. Surgery Animals were anesthetized with intraperitoneal injection of 100 mg/kg ketamine in combination with 20 mg/kg xylazine and then were subjected to stereotaxic surgery (Stoelting, Wood Dale, IL, USA). Guide cannulas were inserted bilaterally into the dorsal hippocampus (CA1 region) and were attached to the skull surface using orthopedic cement. Stereotaxic coordinates used for injection into the rat brain, based on the atlas of Paxinos and Watson, were as follows: anterior–posterior, 3.8 mm from bregma; mediolateral, 2.2 mm from midline; dorsoventral, 2.7 mm from the skull surface (Paxinos and Watson, 1997). After stereotaxic surgery and cannulation, animals remained in their cages for one week as a recovery period during which they were handled daily. Nicotine (1 mg/side) was infused into both sides of the dorsal hippocampus via inserted cannulas 30 min before each training session using a Hamilton syringe. 2.5. Drug treatment 2.5.1. Experiment 1 In this experiment animals were assigned to four groups. While the first group served as the control animals and received 1 cm3/ day of tap water, second, third and fourth groups of animals received 1 cm3 SMV solution (15, 20 and 25 mg/kg/day), by intragastric gavage for 14 consecutive days. One day after the administration of the last dose of vanadium, training was started in MWM. Based on our findings during the MWM training period, we

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selected 25 mg/kg/day as the appropriate dose for studying the interaction between vanadium with nicotine in experiment 2. 2.5.2. Experiment 2 In experiment 2, animals were divided into five groups. One group that underwent stereotaxic surgery and cannula implantation without any injection served as the sham-operated group, while another group which was infused with 1 ml/side normal saline into CA1 region of hippocampus served as the control group. Animals in the nicotine group (third group) received intrahippocampal infusion of 1 mg/side nicotine, 30 min before the beginning of each training session in MWM. The fourth group of animals received 25 mg/kg/day SMV by intragastric gavage for 2 weeks. In last group (fifth group), nicotine (1 mg/side) was administered 30 min before starting of each training day in animals which had been treated previously with SMV (25 mg/kg/day) for 2 weeks. Therefore, technically nicotine administration could be considered rescue therapy for vanadium intoxication in this group. 2.6. Histology Following behavioral evaluations, animals were randomly decapitated and their brains were removed. To check the cannula and needle placement in the CA1 area, histological examinations were done. For this purpose, 100-mm thick brain sections were taken, mounted on slides and stained with cresyl violet. To make sure the sites were in the dorsal hippocampus, the sections were then examined under a light microscope to find the cannulas placements and sites of infusion. The location of the cannula in the CA1 region of the hippocampus was shown in a representative animal (Fig. 1).

followed by incubation with biotinylated anti-Goat IgG or biotinylated anti-Guinea pig IgG for VAChT and ChAT, respectively (1:500 dilution, Vector Labs, Burlingame, CA, USA) in PBS containing 1% serum for 60 min at room temperature. The sections were subsequently incubated with avidin–biotin complex detection solution (ABC Elite Kit; Vector Laboratories) for 45 min at room temperature. The sections were washed in PBS containing 1% normal goat serum (or horse serum according to the primary antibody) and then incubated with a PBS solution containing 0.05% diaminobenzidine-4HCl (Sigma), 0.3% nickel sulfate and 0.3% hydrogen peroxide for 5–15 min to attain the favorite staining intensity. Staining was stopped by washing the sections three times with PBS; the sections were mounted on slides, air-dried and subjected to microscopic evaluation. All photomicrographs were taken at 10X using an OPTIKA microscope equipped with an Optikam PRO 2 camera (Optika, Italy). 2.8. Quantification of VAChT and ChAT immunostaining To evaluate ChAT and VAChT immunoreactivity, all captured images were analyzed with ImageJ (NIH, USA). The average optical intensity (total intensity/area) of the CA1 region images was calculated for each brain section. The optical density of the background was then subtracted from the optical density of the CA1 area and the normalized values were compared statistically between the control and treated groups. In MSA, the number of VAChT- and ChAT-containing neurons in control and treated animals was determined by counting the cells in all of the brain slices as described previously (Azami et al., 2010; Sharifzadeh et al., 2006). 2.9. Statistical analysis

2.7. Immunohistochemistry For immunostaining brain tissues were obtained from those animals which had been trained previously in MWM. These brains were sectioned and processed according to regular protocols (Karimfar et al., 2009; Woolf et al., 2001). To examine ChAT and VAChT protein expression, sections from MSA and CA1 regions of dorsal hippocampus were selected and immunostained. In brief, sections were blocked with horse serum (HS) for ChAT and normal goat serum (NGS) for VAChT staining. The sections were then incubated with polyclonal antibodies (Millipore, Temecula, CA, USA), against ChAT or VAChT (diluted 1:200) in PBS containing 1% serum (NGS or HS as described) and 0.3% tritonX 100 (TX-100) for 48 h on a shaker at 4 8C. After incubation with primary antibody, sections were washed repeatedly (5 min  5 min) with PBS containing 0.3% TX-100

Fig. 1. The infusion site into the dorsal hippocampus. The figure shows a representative photomicrograph of the infusion site in the rat hippocampal CA1 area with the arrowhead pointing to the infusion cannula tract. CA1, Ammon’s horn region 1; DG, dentate gyrus. Scale bar = 0.5 mm.

One-way analysis of variance (ANOVA) followed by Newman– Keuls multiple comparison post hoc test was used to assess the potential differences in averaged behavioral and molecular scores. For those results in which data were obtained from the hidden platform trials over training days, a two-way ANOVA was chosen for evaluation the effects of SMV doses and training days as two factors and escape latency and traveled distance as repeated measure on each day. A p-value of 0.05 or less was considered statistically significant. 3. Results 3.1. Oral administration of SMV impairs spatial learning in MWM Pre-training administration of SMV (15 mg/kg/day, oral) for a period of 2 weeks did not alter the escape latency and traveled distance significantly compared with control animals (Fig. 2A and B). Again, a notable increase in time and distance for finding the hidden platform was caused by applying the 25 mg/kg/day of SMV (Fig. 2A and B), as compared with the control group (***p < 0.001). The swimming speed was the same in all treated groups (Fig. 2C). Each value represents the mean  SEM of all training sessions. Comparing the same training days of different groups by twoway ANOVA in experiment 1, we found that administration of SMV (25 mg/kg/day) increased escape latency (F(3,176) = 13.36; p < 0.0001) on the first (**p < 0.01), second (*p < 0.05), third (**p < 0.01) and fourth (*p < 0.05) day of training in comparison with respective control groups (Fig. 3A). Moreover, significant increases in traveled distance (F(3,176) = 9; p < 0.0001) were also observed in day 2 (*p < 0.05), day 3 (**p < 0.01) and day 4 (*p < 0.05) compared with control animals (Fig. 3B). The swimming speed was not affected in all treated groups of animals (data not shown).

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Fig. 3. Evaluation of escape latency (A) and traveled distance (B) in the same training day in SMV-treated animals. The figure shows significant increase in escape latency in 25 mg SMV group on the first (**p < 0.01), second (*p < 0.05), third (**p < 0.01) and fourth (*p < 0.05) days of training compared to control group. Considerable increase in traveled distance was seen in 25 mg SMV-treated group on day 2 (*p < 0.05), day 3 (**p < 0.01) and day 4 (*p < 0.05) in comparison with control group (n = 8).

difference in escape latency and traveled distance for finding the visible platform among all treated animals (Fig. 4A and B). 3.3. Interactive effects of oral administration of SMV and bilateral intra-hippocampal infusion of nicotine on time and distance for finding the hidden platform during training trials

Fig. 2. Effects of 2 weeks of oral administration of sodium metavanadate (SMV, 15, 20 and 25 mg/kg/day) on escape latency (A), traveled distance (B) and swimming speed (C) in MWM. The average of all training trials during 4 days was shown in this figure. Each value represents the mean  SEM from all training sessions of 8 rats. ***p < 0.001 significantly different from the control animals.

Bilateral infusions of nicotine (1 mg/side) into the CA1 region of the hippocampus, 30 min before the beginning of each training session, significantly decreased the time (*p < 0.05) and distance (**p < 0.01) for finding the hidden platform compared with control (saline treated) animals (Fig. 5A and B). This finding indicates that intra-hippocampal infusion of nicotine during training days improves spatial learning in the MWM. In addition, pre-treatment of animals with SMV (25 mg/kg/day, 2 weeks) significantly induced learning deficit in MWM (***p < 0.001). Moreover, combined administration of SMV and nicotine decreased the time (###p < 0.001) and distance (###p < 0.001) for finding the hidden platform significantly in comparison with SMV treated animals (Fig. 5A and B). As expected, sham group did not show any significant alterations compared with control animals (Fig. 5A and B). The swimming speed was not affected in any of the treated rats, representing the lack of motor disturbances (Fig. 5C). The visible performance evaluation did not show any significant changes in spatial learning parameters in any of the treated groups (Fig. 6A and B).

3.2. Assessment of visuo-motor coordination toward a visible platform in MWM after 2 weeks of SMV oral administration

3.4. Interactive effects of oral administration of SMV and bilateral intra-hippocampal infusion of nicotine on cholinergic nerve terminal density in the dorsal hippocampus and cholinergic cell bodies expression in the medial septal area

On day 5, the platform was elevated 1 cm above the water surface and placed at the center of the quadrant opposite to the previously hidden platform in MWM. Evaluation of visuo-motor coordination toward a visible platform showed no significant

Rat brain tissue sections from control animals as well as the animals treated with nicotine, vanadium and nicotine–vanadium combinations were immunostained with anti-ChAT and antiVAChT antibodies. Bilateral infusion of nicotine (1 mg/side),

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Fig. 4. The effect of different doses of oral sodium metavanadate (SMV, 15, 20 and 25 mg/kg/day for 2 weeks) on visuo-motor coordination toward a visible platform in MWM. There is no significant difference in time (A) and distance (B) of finding the visible platform between treated groups and control animals which indicate the lack of visio-motor dysfunctions (n = 8).

significantly increased the expression of ChAT (***p < 0.001) and VAChT (*p < 0.05), in CA1 region of the hippocampus (Fig. 7A and B). Likewise, the number of ChAT (**p < 0.01) and VAChT (***p < 0.001) immunopositive neurons was remarkably enhanced in MSA (Fig. 8A and B). Oral administration of SMV (25 mg/kg/day, 2 weeks) caused significant reduction in the expression of ChAT (***p < 0.001) and VAChT (*p < 0.05), in the CA1 region compared with the control group. Similarly, the number of ChAT and VAChT immunopositive neurons in the MSA was remarkably decreased (***p < 0.001) (Fig. 8A and B). Interestingly, bilateral intrahippocampal infusion of nicotine rescued expression of ChAT (#p < 0.05) and VAChT (#p < 0.05) from toxic effects of oral SMV in CA1 region (Fig. 7A and B). However, in MSA nicotine rescued only ChAT expression (###p < 0.001) from toxic effects of SMV (Fig. 8A and B). 4. Discussion In this study we examined the molecular and behavioral effects of orally administered SMV on spatial learning in the MWM. The protective effects of intrahippocampal infusion of nicotine on SMV-induced learning deficits were also assessed by quantitative measurement of ChAT and VAChT expression in both CA1region of hippocampus and MSA. Our results showed that intrahippocampal infusion of nicotine during training trials was able to attenuate the detrimental effects

Fig. 5. Effects of intra-hippocampal infusion of nicotine (1 mg/side) in SMVpretreated animals (25 mg/kg/day, for 2 weeks) on escape latency (A), traveled distance (B) and swimming speed (C) in MWM. SMV significantly increased the time and distance for finding the hidden platform (***p < 0.001). CA1 intrahippocampal infusions of nicotine (1 mg/side) caused significant decrease in time (*p < 0.05) and distance (**p < 0.01) for finding the hidden platform compared to control group. Bilateral intra-hippocampal infusion of nicotine during training days improved the SMV-induced spatial acquisition impairments in MWM (###p < 0.001). There are no significant differences in the swimming speed among different groups (Fig. 3C). Each value represents the mean  SEM of all training trials in each group (n = 8).

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Fig. 6. Effects of intra-hippocampal infusion of nicotine (1 mg/side) in SMVpretreated animals (25 mg/kg/day, for 2 weeks) on visuo-motor coordination toward a visible platform in MWM. There is no significant difference in time (A) and distance (B) of finding the visible platform between treated groups and control animals (n = 8).

of vanadium on learning, but the swimming speed was not affected in treated groups indicating the lack of motor dysfunction. Moreover, the visible platform test showed no significant changes in escape latency, traveled distance or swimming speed among groups. These results confirmed that vanadium and nicotine exposure did not affect the motivational or visual systems, which contribute to water maze performance. We observed that SMV caused learning deficit in a dosedependent manner. While lower concentrations of SMV did not lead to impairment on acquisition, higher dose of the compound induced significant spatial learning deficits. There are some controversies about the impact of vanadium on learning and memory. While some reports indicate that inhalation of vanadium compounds disrupts spatial memory through cell necrosis and reduction of dendritic spines in the hippocampus (Avila-Costa et al., 2005, 2006; D‘Hooge and De Deyn, 2001), other studies suggest neuroprotective properties for vanadium against ischemic neuronal injury and olfactory bulbectomy-induced neurodegeneration in rodents (Han et al., 2008; Hasegawa et al., 2006). Recently, it has been reported that administration of VO (HB(3,5-Me2pz)3)(3,5-Me2pzH)(SCN)(SCNH)2, a new vanadium derivative, causes spatial memory improvement in alloxaninduced diabetic mice through the activation of MAPK/CREB signaling pathway (Chen et al., 2008). It has been shown that vanadium is able to inhibit protein kinase A at high concentrations (Jelveh et al., 2006). A large body of evidence has indicated that activation of cAMP-PKA pathway has important roles in learning and memory improvement (Abel

Fig. 7. Quantitative analysis of ChAT and VAChT immunoreactivity in the hippocampal CA1 region. Optical density measurements were performed for ChAT and VAChT immunoreactivity in the CA1 region of hippocampus for control animals and treated groups. There was a significant increase in the staining intensity of ChAT (***p < 0.001) and VAChT (*p < 0.05)-containing neurons in nicotine (1 mg/side) treated animals. Oral administration of SMV (25 mg/kg/day, for 2 weeks) reduced ChAT (***p < 0.001) and VAChT (*p < 0.05) immunoreactivity compared with control group. Pretraining intrahippocampal infusion of nicotine (1 mg/side) in SMV-treated animals attenuated SMV-induced decrease of ChAT and VAChT optical density in comparison with the SMV group (#p < 0.05). Each value represents the mean  SEM from brain tissue sections of at least 5 animals in each treated group.

et al., 1997; Bernabeu et al., 1997; Guerra et al., 2011; Quevedo et al., 2004). We have previously reported the time-related effects of PKA on learning and memory processes (Eftekharzadeh et al., 2011). Considering the inhibitory effects of high concentrations of SMV on PKA activity, and the time-related effects of PKA in learning functions, it is reasonable to deduce that the doses and times of assessment are probably involved in SMV-induced spatial learning deficit through PKA inhibition. However, various parameters such as the chemical form and concentration of vanadium compounds, stage of learning and memory evaluation, experimental task as well as the treatment protocol and exposure route are involved in the observed effects of vanadium.

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Fig. 8. Quantitative analysis of ChAT and VAChT immunopositive neurons in the medial septal area. There was a significant increase in the number of ChAT (***p < 0.001) and VAChT (**p < 0.01) immunopositive neurons in animals receiving intra-hippocampal infusion of nicotine (1 mg/side) in comparison with control rats. Oral vanadate (25 mg/kg/day, 2 weeks) treatment significantly reduced ChAT and VAChT immunopositive neurons compared to control group (***p < 0.001). Infusion of nicotine in combination with oral vanadate induced a significant increase in the number of ChAT immunopositive neurons compared to vanadate-treated animals (###p < 0.001). Each value represents the mean  SEM from brain tissue sections of at least 5 animals in each treated group.

We found that vanadium administration decreased ChAT and VAChT expression in MSA and CA1 region of hippocampus. The learning and memory process requires new protein synthesis through cAMP/PKA/CREB signaling pathway (Drain et al., 1991). ChAT and VAChT are among these newly synthesized proteins which play a critical role in cholinergic transmission. In presynaptic nerve terminals, ChAT is responsible for the synthesis of acetylcholine and VAChT is responsible for the transport of acetylcholine into synaptic vesicles (Erickson et al., 1994). In addition, it has been reported that the expression of ChAT and VAChT is co-regulated in cholinergic neuronal system (Berrard et al., 1995). We observed that vanadium administration induced 73.9% and 47.5% reduction of ChAT and VAChT optical density in CA1 region respectively. It is well documented that the administration of PKA II inhibitor, leads to deterioration of spatial memory

which is associated with reduction of ChAT and VAChT proteins expression (Sharifzadeh et al., 2005b). Thus, vanadium by affecting the cAMP/PKA signaling cascade can interact with the expression of cholinergic markers which in turn causes spatial learning deficit. However, other mechanisms such as the up-regulation of acetyl cholinesterase (AChE) expression or down-regulation of cholinergic binding site can be involved in the toxicity of vanadium on the cholinergic system (Danielsson et al., 1983; Ghareeb and Hussen, 2008). Our results showed that nicotine improved vanadium-induced spatial learning deficit by decreasing escape latency and traveled distance to control levels. We also observed that nicotine ameliorated SMV-induced reduction of ChAT and VAChT protein expression. There are reports indicating that nicotine provokes the activity of cAMP-PKA pathway (Della Fazia et al., 1997; Hiremagalur and Sabban, 1995; Sharifzadeh et al., 2006). In addition, it is established that concurrent administration of nicotine and dibutyryl cAMP as a cell permeable cAMP agonist improved spatial memory synergistically which was associated with increased expression of ChAT and VAChT (Azami et al., 2010; Sharifzadeh et al., 2005c, 2007). Furthermore, nicotine stimulates the release of a variety of neurotransmitters including acetylcholine which may contribute to improvement of cognitive functions (Hefco et al., 2003; Levin and Simon, 1998; Singer et al., 2004; Wonnacott et al., 1990). Therefore, nicotine via interaction with nAChRs and stimulation of cAMP/PKA signaling pathway can improve learning deficit induced by SMV as indicated by an increase in ChAT and VAChT expression. However, it has not been shown in the present studies that alterations of cholinergic markers expression are responsible for the behavioral effects. The behavioral and neurochemical changes may both result from nicotinic receptor activation induced by nicotine infusion. In attempting to account for the effects of nicotine administration in SMV-pretreated animals, it is important to consider that the behavioral results do not necessarily argue only for a cholinergic mechanism. In other words, the blockade of the vanadium effects on escape latency and traveled distance could be due to a simple addition effect where combination of two drugs leads to cancellation of their functions. Taken together, the present findings from behavioral and molecular analyses demonstrated that alterations of cholinergic signal transduction pathways are involved in learning impairment after vanadium exposure. Moreover, we provided information that neuroprotective effect of nicotine against sodium metavanadate neurotoxicity could be associated with the improvement of learning capabilities and cholinergic markers expressions. However, given the complexity of mechanisms involved in memory formation, more research is needed to fully elucidate the exact mechanism of vanadium effects on learning processes. 5. Conclusion We demonstrated that oral administration of sodium metavanadate solution (25 mg/kg/day, 2 weeks) significantly impaired spatial learning. In addition, bilateral intrahippocampal infusion of nicotine decreased the harmful effects of vanadium on acquisition. According to these results, the neuroprotective effect of nicotine on acquisition impairment induced by sodium metavanadate could be associated with increase expression of ChAT and VAChT cholinergic markers in CA1 region of the hippocampus and medial septal area. Conflict of interest The authors declare that there are no conflicts of interest.

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Acknowledgements This work was supported by grants from Tehran University of Medical Sciences. The authors would like to express their deepest gratitude to Mr. Ali Kazemi for his technical assistance as well as Dr. Maria Rosa Avila-Costa for her excellent help in experimental design. References Abel T, Nguyen PV, Barad M, Deuel TAS, Kandel ER, Bourtchouladze R. Genetic demonstration of a role for PKA in the late phase of LTP and in hippocampusbased long-term memory. Cell 1997;88:615–26. Afeseh Ngwa H, Kanthasamy A, Anantharam V, Song C, Witte T, Houk R, et al. Vanadium induces dopaminergic neurotoxicity via protein kinase Cdelta dependent oxidative signaling mechanisms: relevance to etiopathogenesis of Parkinson’s disease. Toxicology and Applied Pharmacology 2009;240:273–85. al-Bayati MA, Giri SN, Raabe OG, Rosenblatt LS, Shifrine M. Time and dose-response study of the effects of vanadate on rats: morphological and biochemical changes in organs. Journal of Environmental Pathology Toxicology & Oncology 1989;9:435–55. Amorim FA, Welz B, Costa AC, Lepri FG, Vale MG, Ferreira SL, et al. Determination of vanadium in petroleum and petroleum products using atomic spectrometric techniques. Talanta 2007;72:349–59. Avila-Costa MR, Colin-Barenque L, Zepeda-Rodriguez A, Antuna SB, Saldivar OL, Espejel-Maya G, et al. Ependymal epithelium disruption after vanadium pentoxide inhalation. A mice experimental model. Neuroscience Letters 2005;381:21–5. Avila-Costa MR, Fortoul TI, Nio-Cabrera G, Coin-Barenque L, Bizarro-Nevares P, Gutie´rrez-Valdez AL, et al. Hippocampal cell alterations induced by the inhalation of vanadium pentoxide (V2O5) promote memory deterioration. Neurotoxicology 2006;27:1007–12. Azami K, Etminani M, Tabrizian K, Salar F, Belaran M, Hosseini A, et al. The quantitative evaluation of cholinergic markers in spatial memory improvement induced by nicotine-bucladesine combination in rats. European Journal of Pharmacology 2010;636:102–7. Baulieu E-E, Robel P. Neurosteroids. A new brain function? The Journal of Steroid Biochemistry and Molecular Biology 1990;37:395–403. Bernabeu R, Bevilaqua L, Ardenghi P, Bromberg E, Schmitz P, Bianchin M, et al. Involvement of hippocampal cAMP/cAMP-dependent protein kinase signaling pathways in a late memory consolidation phase of aversively motivated learning in rats. Proceedings of the National Academy of Sciences of the United States of America 1997;94:7041–6. Berrard S, Varoqui H, Cervini R, Israe¨l M, Mallet J, Diebler M-F. Coregulation of two embedded gene products, choline acetyltransferase and the vesicular acetylcholine transporter. Journal of Neurochemistry 1995;65:939–42. Berse B, Blusztajn JK. Coordinated Up-regulation of Choline Acetyltransferase and Vesicular Acetylcholine Transporter Gene Expression by the Retinoic Acid Receptor, cAMP, and Leukemia Inhibitory Factor/Ciliary Neurotrophic Factor Signaling Pathways in a Murine Septal Cell Line. Journal of Biological Chemistry 1995;270:22101–4. Bhanot S, McNeill JH. Vanadyl sulfate lowers plasma insulin and blood pressure in spontaneously hypertensive rats. Hypertension 1994;24:625–32. Broadbent NJ, Squire LR, Clark RE, Broadbent NJ, Squire LR, Clark RE. Spatial memory, recognition memory, and the hippocampus. Proceedings of the National Academy of Sciences of the United States of America 2004;101:14515–20. Calderon-Garciduenas L, Azzarelli B, Acuna H, Garcia R, Gambling TM, Osnaya N, et al. Air pollution and brain damage. Toxicologic Pathology 2002;30:373–89. Cam MC, Faun J, McNeill JH. Concentration-dependent glucose-lowering effects of oral vanadyl are maintained following treatment withdrawal in streptozotocin-diabetic rats. Metabolism Clinical & Experimental 1995;44:332–9. Chen DD, Wang L, Liu Y, Che J, Zou W. Vanadium improves the spatial learning and memory by activation of caveolin-MAPK-CREB pathway in diabetic mice. Cell Biology International 2008;32:S53–60. Cohen N, Halberstam M, Shlimovich P, Chang CJ, Shamoon H, Rossetti L. Oral vanadyl sulfate improves hepatic and peripheral insulin sensitivity in patients with noninsulin-dependent diabetes mellitus. Journal of Clinical Investigation 1995;95:2501–9. Cruz TF, Morgan A, Min W. In vitro and in vivo antineoplastic effects of orthovanadate. Molecular & Cellular Biochemistry 1995;153:161–6. D‘Hooge R, De Deyn PP. Applications of the Morris water maze in the study of learning and memory. Brain Research – Brain Research Reviews 2001;36:60–90. Danielsson E, Unde´n A, Bartfai T. Orthovanadate induces loss of muscarinic cholinergic binding sites. Biochemical and Biophysical Research Communications 1983;110:567–72. Decker MW, Brioni JD, Bannon AW, Arneric SP. Diversity of neuronal nicotinic acetylcholine receptors: lessons from behavior and implications for cns therapeutics. Life Sciences 1995;56:545–70. Decker MW, Majchrzak MJ, Anderson DJ. Effects of nicotine on spatial memory deficits in rats with septal lesions. Brain Research 1992;572:281–5. Della Fazia MA, Servillo G, Sassone-Corsi P. Cyclic AMP signalling and cellular proliferation: regulation of CREB and CREM. FEBS Letters 1997;410:22–4. Drain P, Folkers E, Quinn WG. cAMP-dependent protein kinase and the disruption of learning in transgenic flies. Neuron 1991;6:71–82.

51

Eftekharzadeh B, Ramin M, Khodagholi F, Moradi S, Tabrizian K, Sharif R, et al. Inhibition of PKA attenuates memory deficits induced by [beta]-amyloid (1-42), and decreases oxidative stress and NF-[kappa]B transcription factors. Behavioural Brain Research 2011;226:301–8. Erickson JD, Varoqui H, Sch¤fer MK, Modi W, Diebler MF, Weihe E, et al. Functional identification of a vesicular acetylcholine transporter and its expression from a cholinergic gene locus. Journal of Biological Chemistry 1994;269:21929–32. Fortoul TI, Bizarro-Nevares P, Acevedo-Nava S, Pinon-Zarate G, Rodriguez-Lara. ColinBarenque L, et al. Ultrastructural findings in murine seminiferous tubules as a consequence of subchronic vanadium pentoxide inhalation. Reproductive Toxicology 2007;23:588–92. Fortoul TI, Quan-Torres A, Sanchez I, Lopez IE, Bizarro P, Mendoza ML, et al. Vanadium in ambient air: concentrations in lung tissue from autopsies of Mexico City Residents in the 1960 and 1990. Archives of Environmental Health September/ October 2002;57:446–9. Friberg L. Handbook of the toxicology of metals. 2nd ed. Amsterdam: Elsevier; 1986. Garcia GB, Biancardi ME, Quiroga AD, Garcia GB, Biancardi ME, Quiroga AD. Vanadium (V)-induced neurotoxicity in the rat central nervous system: a histo-immunohistochemical study. Drug & Chemical Toxicology 2005;28:329–44. Ghareeb DA, Hussen HM. Vanadium improves brain acetylcholinesterase activity on early stage alloxan-diabetic rats. Neuroscience Letters 2008;436:44–7. Gonzalez-Villalva A, Fortoul TI, Avila-Costa MR, Piian-Zarate G, Rodriguez-Lara V, Martinez-Levy G, et al. Thrombocytosis induced in mice after subacute and subchronic V2O5 inhalation. Toxicology and Industrial Health 2006;22:113–6. Grigoryan GA, Mitchell SN, Hodges H, Sinden JD, Gray JA. Are the cognitive-enhancing effects of nicotine in the rat with lesions to the forebrain cholinergic projection system mediated by an interaction with the noradrenergic system? Pharmacology Biochemistry and Behavior 1994;49:511–21. Guerra GP, Mello CF, Bochi GV, Pazini AM, Fachinetto R, Dutra RC, et al. Hippocampal PKA/CREB pathway is involved in the improvement of memory induced by spermidine in rats. Neurobiology of Learning and Memory 2011;96:324–32. Han F, Shioda N, Moriguchi S, Qin ZH, Fukunaga K. The vanadium (IV) compound rescues septo-hippocampal cholinergic neurons from neurodegeneration in olfactory bulbectomized mice. Neuroscience 2008;151:671–9. Haroutunian V, Barnes E, Davis KL. Cholinergic modulation of memory in rats. Psychopharmacology 1985;87:266–71. Hasegawa Y, Morioka M, Hasegawa S, Matsumoto J, Kawano T, Kai Y, et al. Therapeutic time window and dose dependence of neuroprotective effects of sodium orthovanadate following transient middle cerebral artery occlusion in rats. Journal of Pharmacology and Experimental Therapeutics 2006;317:875–81. Hefco V, Yamada K, Hefco A, Hritcu L, Tiron A, Olariu A, et al. Effects of nicotine on memory impairment induced by blockade of muscarinic, nicotinic and dopamine D2 receptors in rats. European Journal of Pharmacology 2003;474:227–32. Heyliger CE, Tahiliani AG, McNeill JH. Effect of vanadate on elevated blood glucose and depressed cardiac performance of diabetic rats. Science 1985;227:1474–7. Hiramatsu M, Satoh H, Kameyama T, Nabeshima T. Nootropic effect of nicotine on carbon monoxide (CO)-induced delayed amnesia in mice. Psychopharmacology 1994;116:33–9. Hiramatsu M, Yamatsu T, Kameyama T, Nabeshima T. Effects of repeated administration of ( )-nicotine on AF64A-induced learning and memory impairment in rats. Journal of Neural Transmission 2002;109:361–75. Hiremagalur B, Sabban EL. Nicotine elicits changes in expression of adrenal catecholamine biosynthetic enzymes, neuropeptide Y and immediate early genes by injection but not continuous administration. Molecular Brain Research 1995;32:109–15. Jelveh KA, Zhande R, Brownsey RW. Inhibition of cyclic AMP dependent protein kinase by vanadyl sulfate. Journal of Biological Inorganic Chemistry 2006;11:379–88. Kanna PS, Mahendrakumar CB, Chakraborty T, Hemalatha P, Banerjee P, Chatterjee M, et al. Effect of vanadium on colonic aberrant crypt foci induced in rats by 1,2 dimethyl hydrazine. World Journal of Gastroenterology 2003;9:1020–7. Karimfar MH, Tabrizian K, Azami K, Hosseini-Sharifabad A, Hoseini A, Pourghorban M, et al. Time course effects of lithium administration on spatial memory acquisition and cholinergic marker expression in rats. DARU Journal of Pharmaceutical Sciences 2009;17:113–23. Kaul S, Kanthasamy A, Kitazawa M, Anantharam V, Kanthasamy AG, Kaul S, et al. Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1-methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: relevance to oxidative stress in dopaminergic degeneration. European Journal of Neuroscience 2003;18:1387–401. Kim JS, Levin ED. Nicotinic, muscarinic and dopaminergic actions in the ventral hippocampus and the nucleus accumbens: effects on spatial working memory in rats. Brain Research 1996;725:231–40. Levin ED, Simon BB. Nicotinic acetylcholine involvement in cognitive function in animals. Psychopharmacology 1998;138:217–30. Levy BSMD, Hoffman LMD, Gottsegen S, Boilermakers’ Bronchitis. Respiratory tract irritation associated with vanadium pentoxide exposure during oil-to-coal conversion of a power plant. Journal of Occupational Medicine 1984;26:567–70. Mao X, Zhang L, Xia Q, Sun Z, Zhao X, Cai H, et al. Vanadium-enriched chickpea sprout ameliorated hyperglycemia and impaired memory in streptozotocin-induced diabetes rats. BioMetals 2008;21:563–70. Markowska AL, Long JM, Johnson CT, Olton DS. Variable-interval probe test as a tool for repeated measurements of spatial memory in the water maze. Behavioral Neuroscience 1993;107:627–32. McNeilly JD, Heal MR, Beverland IJ, Howe A, Gibson MD, Hibbs LR, et al. Soluble transition metals cause the pro-inflammatory effects of welding fumes in vitro. Toxicology & Applied Pharmacology 2004;196:95–107.

52

K. Azami et al. / NeuroToxicology 33 (2012) 44–52

Olton DS, Papas BC. Spatial memory and hippocampal function. Neuropsychologia 1979;17:669–82. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. New York: Academic Press; 1997. Pugazhenthi S, Hussain A, Yu B, Brownsey RW, Angel JF, Khandelwal RL. Vanadate induces normolipidemia and a reduction in the levels of hepatic lipogenic enzymes in obese Zucker rat. Molecular and Cellular Biochemistry 1995;153:211–5. Puma C, Deschaux O, Molimard R, Bizot J-C. Nicotine improves memory in an object recognition task in rats. European Neuropsychopharmacology 1999;9:323–7. Quevedo J, Vianna MRM, Martins MR, Barichello T, Medina JH, Roesler R, et al. Protein synthesis, PKA, and MAP kinase are differentially involved in short- and long-term memory in rats. Behavioural Brain Research 2004;154:339–43. Robert B. Vanadium: how market developments affect the titanium industry. In: Strategic Minerals Corporation, Titanium 2006, International Titanium Association Conference. San Diego, California; 2006. Sanchez DJ, Colomina MT, Domingo JL. Effects of vanadium on activity and learning in rats. Physiology & Behavior 1998;63:345–50. Sansone M, Castellano C, Battaglia M, Ammassari-Teule M. Effects of oxiracetam– nicotine combinations on active and passive avoidance learning in mice. Pharmacology Biochemistry and Behavior 1991;39:197–200. Sharifzadeh M, Naghdi N, Khosrovani S, Ostad SN, Sharifzadeh K, Roghani A. Post-training intrahippocampal infusion of the COX 2 inhibitor celecoxib impaired spatial memory retention in rats. European Journal of Pharmacology 2005a;511:159–66. Sharifzadeh M, Sharifzadeh K, Naghdi N, Ghahremani MH, Roghani A. Posttraining intrahippocampal infusion of a protein kinase AII inhibitor impairs spatial memory retention in rats. Journal of Neuroscience Research 2005b;79:392–400. Sharifzadeh M, Tavasoli M, Naghdi N, Ghanbari A, Amini M, Roghani A. Post-training intrahippocampal infusion of nicotine prevents spatial memory retention deficits induced by the cyclo-oxygenase-2-specific inhibitor celecoxib in rats. Journal of Neurochemistry 2005c;95:1078–90. Sharifzadeh M, Tavasoli M, Soodi M, Mohammadi-Eraghi S, Ghahremani MH, Roghani A. A time course analysis of cyclooxygenase-2 suggests a role in spatial memory retrieval in rats. Neuroscience Research 2006;54:171–9. Sharifzadeh M, Zamanian A-R, Gholizadeh S, Tabrizian K, Etminani M, Khalaj S, et al. Post-training intrahippocampal infusion of nicotine-bucladesine combination causes a synergistic enhancement effect on spatial memory retention in rats. European Journal of Pharmacology 2007;562:212–20. Singer S, Rossi S, Verzosa S, Hashim A, Lonow R, Cooper T, et al. Nicotine-induced changes in neurotransmitter levels in brain areas associated with cognitive function. Neurochemical Research 2004;29:1779–92.

Stolerman IP, Mirza NR, Shoaib M. Nicotine psychopharmacology: addiction, cognition and neuroadaptation. Medicinal Research Reviews 1995;15:47–72. Tabrizian K, Najafi S, Belaran M, Hosseini-Sharifabad A, Azami K, Hosseini A, et al. Effects of Selective iNOS Inhibitor on Spatial Memory in Recovered and Nonrecovered Ketamine Induced-anesthesia in Wistar Rats. Iranian Journal of Pharmaceutical Research 2010;9:313–20. Vitolo OV, Sant‘Angelo A, Costanzo V, Battaglia F, Arancio O, Shelanski M. Amyloid betapeptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling. Proceedings of the National Academy of Sciences of the United States of America 2002;99:13217–21. Whitehouse PJ, Kalaria RN. Nicotinic receptors and neurodegenerative dementing diseases: basic research and clinical implications. Alzheimer Disease & Associated Disorders 1995;9:3–5. WHO. Vanadium pentoxide and other inorganic vanadium compounds. Concise International Chemical Assessment documents, Document 29, 2001. Wonnacott S, Drasdo A, Sanderson E, Rowell P. Presynaptic nicotinic receptors and the modulation of transmitter release. Ciba Foundation Symposium 1990;152:87–101 discussion 2–5. Woodin MA, Hauser R, Liu Y, Smith TJ, Siegel PD, Lewis DM, et al. Molecular markers of acute upper airway inflammation in workers exposed to fuel-oil ash. American Journal of Respiratory & Critical Care Medicine 1998;158:182–7. Woodin MA, Liu Y, Neuberg D, Hauser R, Smith TJ, Christiani DC. Acute respiratory symptoms in workers exposed to vanadium-rich fuel-oil ash. American Journal of Industrial Medicine 2000;37:353–63. Woodin MA, Liu Y, Hauser R, Smith T, Christiani D. Pulmonary function in workers exposed to low levels of fuel-oil ash. Journal of Occupational & Environmental Medicine 1999;41:973–80. Woolf NJ, Milov AM, Schweitzer ES, Roghani A. Elevation of nerve growth factor and antisense knockdown of TrkA receptor during contextual memory consolidation. Journal of Neuroscience 2001;21:1047–55. Zaporowska H, Wasilewski W. Some selected peripheral blood and haemopoietic system indices in wistar rats with chronic vanadium intoxication. Comparative Biochemistry and Physiology Part C Comparative Pharmacology 1989;93: 175–80. Zarrindast M-R, Sadegh M, Shafaghi B. Effects of nicotine on memory retrieval in mice. European Journal of Pharmacology 1996;295:1–6. Zhou DL, Feng CY, Lan YJ, Wang ZM, Huang S, Wang MZ, et al. Paired-control study on the effect of vanadium on neurobehavioral functions. Sichuan da Xue Xue Bao Yi Xue Ban/Journal of Sichuan University Medical Science Edition 2007;38:468–70.