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Current Trends in Biotechnology and PharmacyVol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1195Locomotory Activit: Locomotor activity of therat was studied with Opto-Varimex mini(Columbus Instruments, USA). The data wastaken as total duration 30minutes to each animalduring the 5 minutes session interval.Exploratory Behaviour: Exploratory behaviourwas evaluated in the hole board. The apparatuswas an open-field arena with four equally spacedholes (3 cm in diameter) in the floor. Each ratwas placed individually in the centre of the arenafor 5 min, during which we recorded head-dipcount and head-dipping duration, in seconds. Ahead dip was scored if both eyes disappearedinto the hole. Head-dipping duration data areexpressed as total duration during the 5-minsession. The results for head dip are expressedas number of counts, and for head-dippingduration in seconds.Morris water maze: Spatial discriminationlearning not only involves place learning, whichis learning a position in space which in this caseis the position of the hidden platform in the Morriswater escape task, but also involves non-spatialcomponents like procedural learning (such aslearning to search for an escape platform) andvisual or other sensorimotor processes togetherwith possible motivational/emotional processesnecessary for executing the task. After acquisitionof the spatial water escape task, a probe trial canreveal whether the rats have actually learned theposition of the platform. Furthermore, a spatialdiscrimination reversal task after the acquisitionof the spatial task, measures mainly placelearning because the rats are already familiarwith the procedural component.The water maze is a circular water tankmeasuring 1.85 m in diameter and 0.7 m deepconstructed according to a basic design similar tothat of Morris (16). Four points along thecircumference of the water tank were designatedarbitrarily North (N), South (S), East (E), and West(W), thus dividing the maze into four quadrants.The pool was filled to a depth of 30 cm with watermade opaque with white, non toxic water-basedpaint. A circular submerged platform (diameter12.5 cm) remained below the surface of water.All parameters involving time and distance aremeasured in seconds. Testing was carried outon five consecutive days. Control, Pb+Mn treatedand Ca 2+ supplemented rats were subjected towater maze learning tasks.Biochemical StudiesPreparation of mitochondrial fraction:Mitochondrial fractions were prepared byfollowing the method of Lai and Clark (17) byhomogenizing in 5 volumes (w/v) of Sucrose-EDTA-Tris buffer (SET) buffer (0.25 M sucrose,10 mM Tris-HCl, and 1 mM EDTA, pH 7.4). Thehomogenate was first centrifuged at 800 g for 10min at 4°C, and then the supernatant wascentrifuged at 10,000g for 20 min. Then the pelletof mitochondrial fraction was suspended in SETbuffer.Estimation of AChE: The specific activity ofAChE was determined as described by Ellmanet al., (18). The reaction mixture contained 3.0ml of 0.1M phosphate buffer (pH 8.0), 20µl of0.075 M acetylthiocholine iodide and 100 µl of0.01 M 5, 5-dithiobis 2-nitrobenzoic acid. Thereaction was initiated with the addition of 100 µlof crude homogenate. The contents wereincubated for 30min at room temperature and thecolor absorbance was measured at 412nm inspectrophotometer (Hitachi, Model U-2000). Theenzyme activity was expressed as µ moles of AChhydrolyzed /mg protein/h.Estimation of Adenosine Triphosphatase(ATPase) activity: Na + K + and Mg 2+ ATPaseactivities in the tissues were estimated followingthe method of Tirri et al., (19). Briefly, 1%homogenates of the tissues were prepared in 0.25M ice cold sucrose solution. Homogenates wereCalcium protection against lead and manganese toxicity
Current Trends in Biotechnology and PharmacyVol. 5 (2) 1193-1205 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1196divided into two parts. One part was centrifugedat 1400g and the supernatant thus obtained wasused as an enzyme source for Mg 2+ ATPase, whilethe other part of the homogenate was used forthe estimation of the total ATPase.Mg 2+ ATPase: The reaction mixture forMg 2+ ATPase assay contained 0.5 ml of tris buffer(0.13 M; pH 7.4), 0.4 ml of substrate ATP, 0.5 mlof Magnesium chloride (0.05 M MgCl 2) and 0.2ml of mitochondrial fraction (enzyme source). Thecontents were incubated at 37° C for 15 minutesand the reaction was stopped by the addition of10% TCA. Zero time controls were maintainedby adding TCA prior to the addition ofhomogenate/mitochondrial fraction. The contentswere centrifuged at 1000g for 15 minutes andthe inorganic phosphate was estimated in thesupernatant fraction following the method of Fiskeand Subbarow (20).Na + K + ATPase: 1% (W/V) homogenate alreadyset apart was used for the total ATPase assay.The reaction mixture in a final volume of 2.6 mlcontained, 0.5 ml of Tris buffer (0.13 M; pH 7.4),0.4 ml of substrate ATP, 0.5 ml MgCl 2(0.05 M),0.5 ml potassium chloride (KCl, 0.05 M), 0.5 mlof sodium chloride (NaCl, 0.05 M) and 0.2 ml ofcrude homogenate/ mitochondrial fraction(enzyme source). The contents were incubatedat 37° C for 15 minutes and the reaction wasarrested by the addition of 1.5 ml of 10% TCAprior to the addition of homogenate. The contentswere centrifuged and the inorganic phosphate wasestimated in the supernatant fraction (Na +K + ATPase = Total ATPase - Mg 2+ ATPase).Estimation of protein content: Protein contentof the tissues was estimated by the method ofLowry et al. (21).Analysis of Data: The data was subjected tostatistical analysis (ANOVA and t-test asapplicable) using standard statistical procedures.ResultsOpen field behavior:The results presented inFig.1 show decreased locomotor behavior in allresponses (crossings, rearings, sniffings,groomings) of the open-field in Pb+Mn exposedrats compared to controls. The control rats showedhigher locomotor activity (111.6 crossings, 14.2rearings, 19.2 sniffings, 19.2 groomings) comparedto Pb+Mn treated rats (31.7 crossings, 3.1rearings, 3.7 sniffings, 4.3 groomings). However,the addition of calcium to Pb+Mn, showed amarginal reversal in the alterations observed inthe locomotor behavior (46.88 crossings, 4.33rearings, 6.77 sniffings, 7.33 groomings) ofPb+Mn treated rats.Locomotor activity: The results of locomotoractivity (Fig.2) showed a decrease in the totalnumber of movements in the Pb+Mn treated ratsthan the control rats. The administration ofcalcium along with the Pb+Mn, showed increasein the locomotory activity than Pb+Mn exposedrats.Fig.1. Open-field behavior of control, Pb+Mn exposedand Ca 2+ supplemented rats. Rats were exposed to0.2% lead acetate through drinking water and Mn (2.5mg/kg) intraperitonially daily for a period of 21 days.Calcium (0.02%) was added in 0.2% Pb water. Each barrepresents mean ± SD (n = 6). The values marked withasterisk (*) are significantly different from controls atp
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