high fat diet influences spatial learning and brain cholinergic...

Current Trends in Biotechnology and PharmacyVol. 8 (1) 18-28 January 2014, ISSN 0973-8916 (Print), 2230-7303 (Online)

AbstractDiets rich in high fats have shown to exert

detrimental effects on brain chemistry andcognitive functions. The present study wasdesigned to examine the high fat (HF) dietinduced age related perturbations in cholinergicand antioxidant systems of brain and associateddysfunctions in cognitive behavior of rats. Wistaralbino rats (2 and 6 months) were fed with controland HF diet separately for a period of 8 weeks. Itis evident that no significant changes wereobserved in body weight except in 7th and 8th weekafter consumption of HF diet. However,significant increased levels in total cholesteroland triglycerides were recorded in both 4 and 8months age groups of HF diet fed rats. Resultsalso showed decreased synaptosomalacetylcholinesterase (AChE) and, mitochondrialsuperoxide dismutase (SOD) activities where asMDA levels increased in the cortex, hippocampusand cerebellum regions of both age groupsfollowing consumption of HF diet. These HF dietinduced alterations in cholinergic and antioxidantsystems were greater in cortex and morepronounced at 8 months age rats. Spatiallearning, memory and exploratory behaviors alsodecreased in 4 and 8months age groups of HFdiet fed rats. In conclusion, our results suggestthat HF diet-induces impairments in spatiallearning and alterations in brain cholinergic andantioxidant systems in age dependent manner.

Keywords: high fat diet, cholinergic system,antioxidant system, behavior, brain.

IntroductionThe consumption of fat-rich diets has

increased significantly over the past decade andcontributing to the world wide epidemic obesity(1). The high-fat (HF) diet consumption has beenlinked to development of chronic diseasesincluding diabetes, hypertension, cardiovasculardiseases and also cognitive function deficits (2-4). Epidemiological studies have established anassociation between high intake of fats andincreased risk of developing cognitive impairment(5,6). Different experimental studies also showedthat consumption of HF diet is associated withincreased vulnerability of cholinergic system anddeficits in cognitive functions (7,8).

Recent studies showed altered braincholinergic system and deficits in memory andlearning, exploratory behavior after consumptionof HF diets (9,10). Experimental andepidemiological studies have also showed thatobesity and metabolic dysfunctions are highlyassociated with increased oxidative stress(11,12). Rinder et al., (13) and Dhibi et al., (14)reported that HF diet increased the lipidperoxidation and alterations in the markers ofapoptosis by impairment in the activities ofantioxidant enzymes superoxide dismutase,(SOD) catalase (CAT) and glutathioneperoxidase (GPx) in the tissues of rat. Most ofthe available research studies on the impact ofHF diet concentrated on old age rats and lessattention has been focused on age-relatedimpairments in cognitive functions. Therefore,

High Fat Diet Influences Spatial Learning and BrainCholinergic and Antioxidant Systems in Male Rats

Usha Rani, M.1*, Chand Basha, D.2 and Jamuna, D.1

1Department of Psychology, Sri Venkateswara University, Tirupati, India.2Department of Zoology, Sri Venkateswara University, Tirupati, India.

*For correspondence - [email protected]

Spatial Learning and Brain Cholinergic and Antioxidant Systems

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this study was undertaken to determine the HFdiet induced age related impairments in braincholinergic and antioxidant systems andassociated dysfunctions in spatial learning andexploratory behavior of rats.

Materials and MethodsFor the present study, 2 months and 6

months old male Wistar albino rats were fed witha control and high fat diets separately for a periodof 8 weeks.

Diet: The animals were fed in the laboratory withhigh fat and control diets supplied by NationalInstitute of Nutrition (NIN), Hyderabad, India andwater ad libitum. The High fat diet contained thefollowing ingredients: Casein-2.736 Kg, L-cystine-0.024 Kg, Starch - 1.376 Kg, Sucrose - 1.376Kg, Cellulose-0.40 Kg, Ground Nut oil-0.200 Kg,Tallow- 1.52 Kg, Mineral Mix (AIN 93 grade) -0.28 Kg, Vitamin Mix (AIN 93 grade) - 0.08 Kg.The animals were housed in clear plastic cageswith hardwood bedding in a room maintained at28o ± 2o C and relative humidity 60 ± 10% with a12 hour day/night cycle. Animal experiments werecarried out in accordance with NIH and ICMR(India) guidelines. The approval to conduct theexperiments was obtained from the Institutionalanimal ethical clearance committee, S. V.University. Both age groups of rats were weighedevery week for the period of two months.

Serum total cholesterol and triglycerides:Total cholesterol and triglycerides were estimatedby using commercially available kits (biosystems) with auto analyzer in serum of controland high fat diet fed rats at 4 and 8 months.

Isolation of mitochondrial and synaptosomalfractions: Synaptosomes were isolated frombrain homogenates using Ficoll-sucrosegradients (15). The cerebral cortex, cerebellumand hippocampus were isolated in coldconditions. The tissues were weighed andhomogenized in 10 ml of ice-cold homogenizingbuffer (320 mM sucrose, 10 mM imidazole, 1 mMEDTA) and the volume was brought up to 25 mlwith homogenizing buffer. The homogenates

were centrifuged at 750 × g for 10 min. Thepellets were discarded. The supernatants werecentrifuged at 17,000 × g for 20 min. The pelletswere suspended in 10 ml of 0.32 M sucrose andwere layered on a two-step discontinuous Ficoll–sucrose gradient consisting 13 and 7.5% Ficolland centrifuged at 65,000 × g for 45 min. Themilky layer was formed at the interface of 13 and7.5% Ficoll. The pellet formed at the bottom ofthe centrifuge tube (mitochondrial fraction) wastaken and suspended in homogenizing buffer.The milky layer fraction was diluted with ninevolumes of 0.32 M sucrose and centrifuged again17,000 × g for 30 min. The supernatant wasdiscarded and the pellet (synaptosomal fraction)was suspended in 0.32 M sucrose.

Cholinergic systemEstimation of Acetylcholine (ACh): Theacetylcholine content was estimated by themethod of Metcalf (16) as given by Augustinson(17). The synaptosomal fractions ofhippocampus and cerebellum were placed inboiling water for 5 minutes to terminate the AChEactivity and also to release the bound ACh. Tothe synaptosomal fractions 1 ml of alkalinehydroxylamine hydrochloride followed by 1 ml of50% hydrochloric acid were added. The contentswere mixed thoroughly and centrifuged. To thesupernatant 0.5 ml 0.37 M ferric chloride solutionwas added and the brown colour developed wasread at 540 nm against a reagent blank in aspectrophotometer and expressed asacetylcholine (ACh) content (µ moles of ACh/gmwt.).

Estimation of Acetylcholinesterase activity(AChE): AChE specific activity was determinedfollowing the method of Ellman et al.,(18). Thereaction mixture contained 3.0 ml of phosphatebuffer (pH 8.0), 20 µl of 0.075M acetylthiocholineiodide (substrate) and 100 µl of 0.01 M DTNB(5, 5-Dithiobis-2-Nitrobenzoic acid). The reactionwas initiated with the addition of 100 µl ofsynaptosomal fraction. The contents wereincubated for 30 min at room temperature andthe color absorbance was measured at 412 nmin spectrophotometer.

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Oxidative stress marker enzymesEstimation of Superoxide dismutase (SOD)activity: Measurement of total SOD activity wasperformed according to Misra and Fridovich (19)based on the inhibition of autoxidation ofepinephrine. The total reaction mixture containedof 880 µ l of carbonate buffer, 20 µl of epinephrineand 100 µl of enzyme source and absorbancewas recorded at 480 nm against reagent blank.The enzyme activity was expressed as µ molesof superoxide anion reduced/ mg of protein.

Estimation of MDA levels: The lipid peroxideswere determined by the TBA method of Hiroshiet al.,(20). The tissues were homogenized in1.15% KCl (20% W/V). To 1 ml of tissuehomogenate, 2.5 ml of 20% trichloro acetic acid(TCA) was added and the contents werecentrifuged at 3500g for 10 minutes. Residue wasdissolved in 2.5 ml of 0.05 M sulphuric acid(H2SO4). To this 3 ml of thiobarbituric acid (TBA)was added and the samples were kept in a hotwater bath for 30 minutes. The samples werecooled and malondialdehyde (MDA) wasextracted with 4 ml of n-butanol and read at 530nm in a spectrophotometer against the reagentblank. The results were expressed asmicromoles of MDA formed/mg of tissue/hr.

Estimation of protein content: Protein contentof the tissues was estimated by the method ofLowry et al., (21). To 0.1 ml of synaptosomalfraction, 1ml 10%TCA was added and thesamples were centrifuged at 1000g for 15minutes. Supernatant was discarded and theresidues were dissolved in 1ml of 1N sodiumhydroxide (NaOH). To this, 2 ml of alkaline copperreagent was added followed by 0.2 ml of folin-phenol reagent (1:1folin:H2O). The color wasmeasured at 600 nm in spectrophotometer(Hitachi model U-2000) against a blank. Theprotein content in mitochondrial fraction was alsomeasured with the same procedure. The proteincontent of the tissues was calculated using thestandard graph. The values were expressed asmg protein/gm wet weight of the tissue.

Behavioral Studies

Morris Water Maze: The water maze is a circularwater tank measuring 1.85 m in diameter and0.7 m deep constructed according to a basicdesign similar to that of Morris (22). Four pointsalong the circumference of the water tank aredesignated arbitrarily North (N), South (S), East(E) and West (W), thus dividing the maze intofour quadrants. The pool was filled to a depth of30 cm with water made opaque with white, non-toxic water-based paint. A circular platform(diameter 12.5 cm) is placed 2-3 cm below thesurface of the water. Rats were allowed to swimto the hidden platform and the escape latency(time to find the hidden platform) was noted.Animals were tested for a total of 2 daily trials for5 days. Each trial was separated by 30 minutes.Rats were placed in water facing the wall of thepool and allowed 60 seconds to find the escapeplatform. If the animal reached the escapeplatform within 60 seconds, the escape latencywas recorded and the mouse was allowed to reston the platform for 10 seconds before removal.Each age group was separately tested. In testsof reference memory, the hidden platformremained in the same location throughout eachphase of the experiment. During the acquisitionphase, the platform was placed in the centre ofthe quadrant, 15 cm from the edge of the pool,and it was moved to the opposite quadrant forthe reversal phase. In tests of working memory,each rat was required to find the hidden platformlocated in a new position. The location of thehidden platform was changed daily in a pseudo-randomized fashion such that different rats weretested in all quadrants on a given day, and allrats were trained in each quadrant at least twiceduring the experiment.

Exploratory behavior: Exploratory behavior wasmeasured in a box with a hole board bottom (90cm × 90 cm) containing three equally spacedholes (3 cm in diameter) in the floor. Each ratwas placed in the center of the arena for 5 minduring which time the number of head dips andhead-dipping duration (in seconds) were


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recorded. A head dip was scored if both eyesdisappeared into the hole.

Analysis of Data: The data were subjected toone way analysis of variance (ANOVA) followedby student Newman-Keuls (SNK) post hoc test.The 0.05 level of probability was used as thecriterion for significance.

ResultsIn the present study, analysis of body weight

over the 8 experimental weeks, established nosignificant changes (P >0.05) in body weightexcept in 7th and 8th week of 4 and 8 months agegroups of HF diet fed rats, compared to controls(Fig.1). Total cholesterol and triglycerides levelsin serum were estimated using Bio-system kits.A significant increase was observed in both totalcholesterol and triglyceride levels in both the agegroups of HF diet fed rats compared to controls(Fig. 2 and 3). The specific activity ofsynaptosomal AChE and ACh levels wereestimated in cortex, cerebellum andhippocampus regions at 4 and 8 months agegroups of rats (Fig. 4 and 5). The hippocampusregion exhibited higher levels of AChE activityand ACh levels than cortex and cerebellum (Fig.4 and 5). The specific activity of AChE as wellas ACh levels were decreased in HF diet fed rats(The decrease in AChE was 3.9% and 21.1% incortex, 2.3% and 16% in cerebellum, and 9.4%and 29.1% in hippocampus regions respectivelyof 4 and 8 months age groups of rats where asthe decrease in ACh levels were 25.3% and,33% in cortex, 13.3% and 31% in cerebellumand 9.2% and 21.1% in hippocampus regionsrespectively of 4 and 8 months age groups ofrats) compared to controls. (Fig. 4 and 5).

Further, we evaluated the mitochondrialSOD activity and tissue MDA levels in cortex,cerebellum and hippocampus regions (Fig. 6 and7). The SOD enzyme activity and MDA levelswere increased with age and the highest activityof SOD and MDA levels were observed in cortexthan cerebellum and hippocampus. HF diet fedrats showed decrease in the activity of SOD(cortex: 9.7% in 4 months, 32% in 8 months;cerebellum: 5.4% in 4 months, 20.1% in 8

Fig. 1. Effect of HF diet on body weight of 4 and 8months age groups of rats. Male rats were fed withcontrol and high fat diets separately for a period of 8weeks. Values are mean ± SD of eight observations.The values marked with ‘‘asterisk’’ are significantlydifferent from corresponding controls as evaluatedby the ANOVA followed by student Newman– Keuls(SNK) post hoc test (P < 0.05).

Fig. 2. Effect of HF diet on serum cholesterol levelsin 4 and 8 months age groups of rats. Male rats werefed with control and high fat diets separately for aperiod of 8 weeks. Values are mean ± SD of eightobservations. The values marked with ‘‘asterisk’’ aresignificantly different from corresponding controls asevaluated by the ANOVA followed by studentNewman– Keuls (SNK) post hoc test (P < 0.05).

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Fig.3. Effect of HF diet on serum triglyceride levelsin 4 and 8 months age groups of rats. Male rats werefed with control and high fat diets separately for aperiod of 8 weeks. Values are mean ± SD of eightobservations. The values marked with ‘‘asterisk’’ aresignificantly different from corresponding controls asevaluated by the ANOVA followed by studentNewman– Keuls (SNK) post hoc test (P < 0.05).

Fig.4. Effect of HF diet on synaptosomalacetylcholinestrase (AChE) activity in cortex,cerebellum and hippocampus regions of 4 and 8months age groups of rats. Male rats were fed withcontrol and high fat diets separately for a period of 8weeks. Values are mean ± SD of eight observations.The values marked with ‘‘asterisk’’ are significantlydifferent from corresponding controls as evaluatedby the ANOVA followed by student Newman– Keuls(SNK) post hoc test (P < 0.05).

Fig.5. Effect of HF diet on synaptosomal acetylcholine(ACh) levels in cortex (CC), cerebellum (CB) andhippocampus (HP) regions of 4 and 8 months agegroups of rats. Male rats were fed with control andhigh fat diets separately for a period of 8 weeks.Values are mean ± SD of eight observations. Thevalues marked with ‘‘asterisk’’ are significantlydifferent from corresponding controls as evaluatedby the ANOVA followed by student Newman– Keuls(SNK) post hoc test (P < 0.05).

Fig.6. Effect of HF diet on mitochondrial superoxidedismutase activity (SOD) in cortex (CC), cerebellum(CB) and hippocampus (HP) regions of 4 and 8months age groups of rats. Male rats were fed withcontrol and high fat diets separately for a period of 8weeks. Values are mean ± SD of eight observations.The values marked with ‘‘asterisk’’ are significantlydifferent from corresponding controls as evaluatedby the ANOVA followed by student Newman– Keuls(SNK) post hoc test (P < 0.05).


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Fig.7. Effect of HF diet on MDA levels in cortex (CC),cerebellum (CB) and hippocampus (HP) regions of4 and 8 months age groups of rats. Male rats werefed with control and high fat diets separately for aperiod of 8 weeks. Values are mean ± SD of eightobservations. The values marked with ‘‘asterisk’’ aresignificantly different from corresponding controls asevaluated by the ANOVA followed by studentNewman– Keuls (SNK) post hoc test (P < 0.05).

Fig.8. Effect of HF diet on average escape latency(time in sec) to find the hidden platform duringacquisition, reversal phase tests and working memorytest at 4 months age group rats. Values are mean ±SD of eight observations. The values marked with‘‘asterisk’’ are significantly different from corres-ponding controls as evaluated by the ANOVA followedby student Newman– Keuls (SNK) post hoc test (P <0.05).

Fig.10. Effect of HF diet on exploratory behavior at 4and 8 months age groups of rats. Values are mean ±SD of eight observations. The values marked with‘‘asterisk’’ are significantly different from corres-ponding controls as evaluated by the ANOVA followedby student Newman– Keuls (SNK) post hoc test (P <0.05).

Fig.9. Effect of HF diet on average escape latency(time in sec) to find the hidden platform duringacquisition, reversal phase tests and working memorytest at 8 months age group rats. Values are mean ±SD of eight observations. The values marked with‘‘asterisk’’ are significantly different fromcorresponding controls as evaluated by the ANOVAfollowed by student Newman– Keuls (SNK) post hoctest (P < 0.05).

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months; hippocampus: 6.8% in 4 months, 28.5%in 8 months) and increase in tissue MDA levels(cortex: 8.6% in 4 months, 30.9% in 8 months;cerebellum: 6.4% in 4 months, 20.9% in 8months; hippocampus: 12.2% in 4 months,28.9% in 8 months) in both age groups of rats,compared to controls (Fig. 6 and 7). However,the alterations in specific enzyme activities,neurotransmitter and MDA levels were morepronounced in cortex (P <0.001) thanhippocampus and cerebellum regions andgreater at 8 months (P <0.001) age groups ofrats (Fig.4 to 7) after consumption of HF diet.

Spatial learning and memory were testedin Morris water maze and results from thereference (acquisition and reversal phases) andworking memory tests are summarized in Fig. 8and 9. HF diet fed rats showed increase in theescape latency to find the hidden plat form inacquisition, reversal phases and working memoryof both age groups of rats, compared to control(Fig.8 and 9). The exploratory behavior recordedin three whole board showed shorter head dipduration and lesser head dip counts in HF dietfed rats of both age groups of rats (Fig.10).However, the alterations observed in spatiallearning, memory and exploratory behavior inolder adults were significant (P <0.001) (Fig. 8to 10) and marginal (P >0.05) in young adult rats.

DiscussionThe major findings of the present study are

that, consumption of HF diet can lead toimpairments in spatial learning, memory andbrain cholinergic and antioxidant systems indifferent age groups of rats. HF diet induced anovert obesity characterized by body weight gainand fat deposition in different tissues (1,23). Ratsfed with the HF diet had consistently greater bodyweight than control and the majority of the weightgain occurred from the fourth week in both agegroups of rats after consumption of HF diet.Furthermore, the increase in body weight wasgreater in older adults (8 months) compared toyoung adults after consumption of HF diet. Chenet al.,(24) reported similar body weight gain aftersix weeks in HF diet fed rats.

Diets rich in HF induce alterations in lipidprofiles which consequently increase the serumtotal cholesterol and triglyceride levels in rats (25).Our results also showed significant increase inserum total cholesterol and triglyceride levelsfollowing consumption of HF diet in both agegroups of rats. Roberst et al., (26) reported thatHF diet caused an increase in serum triglyceridesand total cholesterol levels of rats. Differentexperimental studies also showed increasedlevels of lipid profiles in serum and tissues ofrats fed with HF diets (27,28). Interestingly, thepresent study results showed HF diet inducedalterations in serum lipid profiles were greater inolder rats than young adults, suggesting agedependent accommodation to the higher caloricdensity of the HF diet. The link betweendyslipidemia and risk of cognitive impairment ordementia is unclear (29). Indeed, rats fed withhigher amount of dietary fat showed widespreadcognitive deficits on various tasks of learning andmemory (10,30). The results of present studyshowed HF diet fed rats took longer time to findhidden platform and caused impairments inacquisition, reversal phases and working memoryin Morris water maze test at 4 and 8 months agegroups of rats. Molteni et al.,(31) reported thatHF diet consumption increases the deficits inspatial learning and memory of rats. Impairmentsin learning and memory have also been observedin young, adult and aged rats after consumptionof HF diet (10,32). Our findings also showeddecreased exploratory behavior in both agegroups of rats following HF diet consumption.However, HF diet induced impairments in spatiallearning, memory and exploratory behavior weregreater in older age group rats compared toyoung adult rats suggesting age dependenteffects of HF diet. However, the mechanisms ofeffects of dietary fat on cognitive functions arestill not completely understood.

Cognitive dysfunctions are characterizedby a substantial loss of cholinergic system indifferent brain regions of rat (6,33). Morgansternet al.,(10) reported that changes in cognitive andexploratory behaviors induced by consumption


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of HF diet may stem from disturbances incholinergic system in specific brain regions.Another study also reported that HF dietconsumption disturb the brain cholinergic systemin rats (34). However, most of the studies wereconcentrated on whole brain evaluation andmissed the presence of brain region specificelevations in response to HF diet consumption(5,7,35). In this connection, present studyevaluated the brain region specific alterations incholinergic system (ACh and AChE) in twodifferent age groups of rats. Our present findingsdemonstrated that synaptosomal ACh levels andAChE enzyme activity decreased followingconsumption of HF diet in cortex, hippocampusand cerebellum regions at 4 and 8 months agegroups of rats in an age dependent manner.Kaizer et al.,(36) also reported that AChE activityand ACh levels were significantly decreased incortex, hippocampus and hypothalamus regionsfollowing consumption of HF diet. Additionally invitro studies have also suggested that fat intakedecreases the AChE activity and ACh levels (37-39). It is also proved in another study, whichshowed that 8 weeks of HF diet consumptiondepletes the brain ACh levels by modifying theAChE enzyme activity (9,10). However, thedecreased activity of AChE enzyme and levelsof ACh in different brain regions indicate that HFdiet influences the cholinergic system in brainregion specific manner and contributing to theobserved changes in spatial learning, memoryand exploratory behaviors of rats.

The mechanisms by which HF diet inducealterations in cholinergic system are not clearlyknown (40). HF diets increase oxidation of fattyacids through the peroxisomal oxidation pathway,that is associated with increased generation offree radicals and reduce the antioxidant enzymeactivities (11,41). The present study confirms thatHF diet consumption caused decrease inmitochondrial SOD enzyme activity where asMDA levels increased in cortex, cerebellum andhippocampus regions at both age groups of rats.Several recent studies also suggested that HFdiet fed rats showed decrease in the antioxidant

enzyme activities and increase in the MDA levelsin brain of rats (13,42,43). HF diet causedoxidative damage in brain by enhancingperoxidation of membrane lipids due to thegeneration of ROS and decreases the activitylevels of antioxidant enzymes leading to oxidativestress (12,43). Our results clearly showed thatan increased MDA level has been accompaniedby reduction in the activity of SOD enzyme indifferent brain regions of rat. The decreasedantioxidant enzymes activities in HF diet fed ratsare indicative of the oxidative stress and aresponse to the cholinergic system dysfunction.

We have chosen to evaluate brain regionsinstead of whole brain because different regionsmay respond differently to oxidative stress andvulnerability of the cholinergic system (35,40).In the present study, the alterations were morepronounced in cortex compared to hippocampusand cerebellum regions. Previous studies alsosuggested that cortex region is more susceptibleto HF diet consumption (6,35). However, HF dietinduced impairments in cognitive functions andbrain chemistry appears to be age specific(10,44,45). In our study, HF diet induced alterationsin cognitive functions and brain cholinergic andantioxidant systems were marginal in young adultscompared to older adults, perhaps longer HF dietconsumption is required to alter cognitive functionsin young adult rats. In conclusion, the present dataindicate that HF diet influences spatial learning,memory and exploratory behavior and associatedcholinergic and antioxidant systems in a brainregion specific manner and suggest that age isa major factor in determining the detrimentaleffects of high fat diet.

AcknowledgementsThe research work was supported by

Department of Science and Technology (DST),Grant No. SR/WOS-A/LS-157/2010(G).

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high fat diet influences spatial learning and brain cholinergic...

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