Behavioural Brain Research 259 (2014) 60–69

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Brain aging, memory impairment and oxidative stress: A study in Drosophila melanogaster Mohammad Haddadi a , Samaneh Reiszadeh Jahromi a , B.K. Chandrasekhar Sagar b , Rajashekhar K. Patil c , T. Shivanandappa a , S.R. Ramesh a,∗ a

Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore, India Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India c Department of Applied Zoology, Mangalore University, Mangalagangotri, Konaje, India b

h i g h l i g h t s • • • • •

It is the first intensive study on oxidative stress and age-related memory impairment in Drosophila. Old flies showed significant decrease in all forms of olfactory conditioning memory. Aging brain of Drosophila shows decreased antioxidant defense system and increased level of ROS and LPO. Light microscopy of paraffin sections of old flies’ brain showed increased rate of apoptosis. TEM of MBs showed decreased number of synapses and formation of giant mitochondria.

a r t i c l e

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Article history: Received 20 August 2013 Received in revised form 17 October 2013 Accepted 22 October 2013 Available online 30 October 2013 Keywords: Classical olfactory conditioning Aging Age-related memory impairment Oxidative stress

a b s t r a c t Memory impairment during aging is believed to be a consequence of decline in neuronal function and increase in neurodegeneration. Accumulation of oxidative damage and reduction of antioxidant defense system play a key role in organismal aging and functional senescence. In our study, we examined the agerelated memory impairment (AMI) in relation to oxidative stress using Drosophila model. We observed a decline in cognitive function in old flies with respect to both short-lived and consolidated forms of olfactory memory. Light and electron microscopy of mushroom bodies revealed a reduction in the number of synapses and discernible architectural defects in mitochondria. An increase in neuronal apoptosis in Kenyon cells was also evident in aged flies. Biochemical investigations revealed a comparable ageassociated decrease in the activity of antioxidant enzymes such as catalase and superoxide dismutase as well as the GSH level, accompanied by an increase in the level of lipid peroxidation and generation of reactive oxygen species in the brain. There was no significant difference in the activity level of AChE and BChE enzymes between different age groups while immunohistochemical studies showed a significant decrease in the level of ChAT in 50-day-old flies. RNAi-mediated silencing of cat and sod1 genes caused severe memory impairment in 15-day-old flies, whereas, over-expression of cat gene could partially rescue the memory loss in the old flies. We demonstrated that a Drosophila long-lived strain, possessing enhanced activity of antioxidant enzymes and higher rate of resistance to oxidative stress, shows lower extent of AMI compared to normal lifespan strain. Present study provides evidence for involvement of oxidative stress in AMI in Drosophila. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Aging is a natural phenomenon which is associated with functional senescence in most organisms (higher organisms). Free radical theory of aging [1] postulates lifelong accumulation of oxidative damage and dwindling antioxidant defense system.

∗ Corresponding author at: Department of Studies in Zoology, University of Mysore, Manasagangotri, Mysore 570006, Karnataka, India. Tel.: +91 821 2419779. E-mail address: [email protected] (S.R. Ramesh). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.10.036

Induction of oxidative stress via excessive generation of ROS (reactive oxygen species) leads to the formation of oxidatively modified macromolecules including proteins resulting in the impairment of their functions [2]. Brain is rich in polyunsaturated fatty acids and catecholamines as oxidizable substrates and relatively deficient in antioxidant defenses, compared with other organs and, therefore, is highly susceptible to oxidative damage [3]. Oxidative stress is one of the main factors in neuronal death in many neurodegenerative diseases. Several studies on Parkinson’s and Alzheimer’s disease have revealed that the level of natural antioxidant, GSH (glutathione) are lower;

M. Haddadi et al. / Behavioural Brain Research 259 (2014) 60–69

while the levels of the oxidative damage markers, lipid peroxidation (LPO), keto-protein formation and DNA oxidation are higher in these patients [4–7]. Drosophila melanogaster is an excellent model to study the biology of aging and age-related functional impairments due to its well-known short lifespan and numerous age-related functional senescence similarities with human subjects [8]. Since natural populations of Drosophila exhibit polymorphism in the genes involved in olfactory neural circuit [9,10], use of an isogenic laboratory population of flies for experiments enables us to exclude probable genetic variations that might affect behavioral, molecular and biochemical characteristics. Drosophila brain is evolutionarily adapted to save and retrieve olfactory information; a feature that is essential for their survival and reproduction [11,12]. Therefore, learning and memory-related investigations can be conveniently carried out in Drosophila model following classical olfactory conditioning [13]. Further, in the aging Drosophila brain the structural changes in the cells can be correlated with age-related events by microscopic observations. Since Drosophila brain is devoid of blood vessels, abnormal vasculature-related changes can be excluded in studies on age-related memory impairment. [14]. In this study, age-related decline in learning and memory of D. melanogaster has been investigated in relation to biochemical changes related to oxidative stress and correlated with morphological changes in the brain with special reference to the mushroom bodies (MBs), the main site of olfactory memory formation [15].

2. Materials and methods 2.1. Drosophila stocks Wild type D. melanogaster (Oregon K) and long-lived D. melanogaster strains were obtained from Drosophila stock centre, Department of Studies in Zoology, University of Mysore, Mysore and required experimental populations were built up. UAScat-IR (Transformant ID, 6283) and UAS-sod1-IR (Transformant ID, 31552), Vienna RNAi Drosophila Stocks and UAS-cat, (Stock No. 24261) a Bloomington Drosophila stock were obtained from Drosophila stock centre NCBS (National Centre for Biological Sciences) Bangalore, India. MB247-gal4, a mushroom body driver [16], was obtained from the Department of Applied Zoology, Mangalore University, Mangalore, India. The flies were reared and maintained on standard wheat cream agar media supplemented with dry yeast granules at 22 ± 1 ◦ C and 70-80% relative humidity in a vivarium. Adults collected every day from these stocks were aged to obtain different age groups of 5 day old (young), 25 day old (mid-age), and 50 day-old (old) flies.

2.2. Chemicals 4-Methyl Cyclohexanol, 3-Octanol, Pyrogallol, Acetylthiocholine iodide, Butyrylthiocholine iodide, Osmium tetroxide, Propylene oxide, Araldite resin, Thiobarbituric acid (TBA), Dichlorodihydrofluorescein diacetate (DCFH-DA), Acetyl thio Choline Iodide (ATCI), Butyryl thio choline iodide (BTCI), Ethylenediaaminetetraacetic acid (EDTA), GSH, Tetramethoxypropane (TMP) and 5, 5 -dithiobis 2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemical Co. St Louis, CA, USA. TRIzol reagent was procured from Invitrogen, Carlsbad, CA, USA. QPCR cDNA synthesis kit, Brilliant II SYBR Green qPCR Master mix and Stratagene Mx3005P platform were purchased from Agilent Technologies Inc. Santa Clara, CA, USA. All other chemicals used were purchased from Sisco Research Laboratories, Mumbai, India were of the highest purity grade.

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2.3. Behavioral analyses Olfactory learning assay was performed by employing classical olfactory conditioning; wherein for calculation of performance index (PI) a naïve group of flies corresponding to each conditioned group was used [17]. One training cycle consisted of 60 second presentation of 3-octanol (OCT) as the conditioned stimulus (CS+ ) which was paired with twelve 1.5-second pulses of 90 V DC electric shock [unconditioned stimulus (US)] followed by 45 second fresh air as intertrial interval and then 60 second presentation of 4methyl cyclohexanol (MCH) without electric shock (CS- ). Soon after training, the flies were transferred back to the fresh food vials and kept at 25 ◦ C until testing time. The MTM (middle term memory) was measured by conducting a single training cycle wherein trained flies were subjected to memory test at 0, 1, 3 and 7 h after training. To evaluate protein-synthesis-dependent, long term memory (LTM), 10X spaced training cycles with 15 min intervals were used [18] in which memory test on trained flies was conducted 24 h after the last training cycle. To evaluate consolidated anesthesiaresistant memory (ARM), 10X massed training cycles were applied [19]; 2 h after training, the flies were transferred into the glass vials and subjected to cold shock by immersing the vials into the icecold water (4 ◦ C) for a duration of 2 min. After induction of rapid and reversible hypothermic anesthesia, the flies were transferred to food vials and kept at room temperature, while the memory test was conducted 30 min later [20]. Memory retention test was carried out only once using a Plexiglas T-maze in which both CS+ and CSwere introduced simultaneously to opposite arms with the same concentration of chemicals as provided in training step. Each group of trained flies was introduced at the choice point and was allowed to choose between CS+ and CS- over a 120 second time. To obtain a distribution score the number of flies in CS- arm, were quantified and subtracted over total number of flies presented in T-maze. A naïve control group, corresponding to each trained fly group were introduced at T-maze choice point without previous exposure to CS+ , CS- or electric shock and allowed to make choice between the two given odors (CS+ and CS- ). Memory performance indices were calculated by subtracting the naïve score from the trained score and expressed as PI (n = 1). In the present study, the given PI score for each group of flies is the mean of PI scores obtained from 12 replicates, while only naïve scores that were not significantly different from zero (by t-test) were selected for calculation of memory performance [17]. 2.4. Histology The flies were anaesthetized, placed in a fly collar and fixed in Carnoy’s fixative overnight at 4 ◦ C. After fixation, the samples were dehydrated in grades of alcohol (40% to 100%) and then embedded in paraffin. Four-micron thick frontal sections of heads were stained with Hematoxylin-Eosin stain [21]. The stained sections were observed for neuroanatomical details under light microscope (Olympus, CX21FS1). The images captured in the digital camera (Sony Cyber-shot DSC-W730) and were analyzed using Image J software [22]. 2.5. Transmission electron microscopy For ultra structural studies, Drosophila brain were dissected in normal saline, fixed in modified Karnovsky’s fixative prepared in phosphate buffer (pH 7.2) for 24 h, and finally subjected to routine TEM. Briefly, tissues were post-fixed in 1% osmium tetroxide for 1 h and subsequently dehydrated in grades of ethyl alcohol. During dehydration, tissues were en-block stained by treating with 2% uranyl acetate in 95% alcohol for 1 h, followed by absolute alcohol for 1 h and finally cleared in propylene oxide for 30 min.

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Subsequently the tissues were impregnated overnight in 1:1 mixture of propylene oxide: araldite resin, which was changed to 1:3 for 3 h, followed by pure araldite resin for 2-3 hours. Later tissues were embedded in flat moulds with desired orientation and kept in the oven at 60 ◦ C for 48 hours to get polymerized [23]. The plastic blocks were cut using Leica EM UC6 ultramicrotome (M/s. Leica Microsysteme, Austria). Initially, 1-␮m-thick sections collected on plain glass slide were stained using 1% toluidine blue and observed under light microscope in order to locate the neuropile area of Kenyon cells. Later 400-500 A˚ thick ultrathin sections mounted on copper grids were stained using Uranyl acetate and Lead citrate as described by Frasca and Parks [24]. After proper staining, the ultrathin sections were scanned under TEM (Tecnai G2 Spirit Biotwin) at 80 KVA and images of representative areas were captured using MegaView-III digital CCD camera.

Tris-buffer (0.2 M; pH 8.0) and 50 ␮l of DTNB. After 10 min incubation at room temperature, the absorbance was read at 412 nm and expressed as ␮g GSH/mg protein [29].

2.6. Biochemical assays

In this step, Drosophila brains were dissected out on ice and fixed in cold 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) for 2 h. Further, samples were washed thrice with PBST (phosphate buffered saline containing 0.3% Triton X) for 20 min at room temperature. Brains were blocked in 10% normal goat serum in PBST for 60 min at room temperature. These samples were incubated in 1:100 mouse IgG monoclonal anti Drosophila choline acetyltransferase (ChAT) antibody [(4B1, Developmental Studies Hybridoma Bank (DSHB)] in PBST for 55 h at 4 ◦ C, washed for 60 min with PBST at room temperature and incubated in 1:200 goat anti-mouse Alexa Fluor 594 in PBST for 48 h at 4 ◦ C [31,32]. Finally, these brains were washed and mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA).

Samples were prepared by homogenizing the heads of 100 flies in ice-cold sodium-phosphate buffer (0.1 M; pH 8.0), centrifuged at 3000 g for 10 min at 4 ◦ C. The supernatant was filtered before using it as sample for assays. 2.6.1. Catalase and SOD To measure the activity of catalase, 50 ␮l of sample was added to 1 ml reaction mixture containing 3% H2 O2 (8.8 mM) and sodium phosphate buffer (pH 7.4). Absorbance was monitored at 240 nm for 3 min. Enzyme activity was determined by reduction in H2 O2 and expressed as ␮mol of H2 O2 /min/mg protein (␧-H2 O2 44.1 mM−1 cm−1 ) [25]. SOD activity was measured following the method of Marklund and Marklund [26] by monitoring the inhibition of pyrogallol auto-oxidation in a reaction mixture containing 200 ␮l of sample and Tris HCl buffer (0.1 M; pH 8.2). The reaction was initiated by adding 0.5 ml of pyrogallol (2 mM) and monitored for 3 min at 412 nm. Activity of the enzyme was expressed as amount of protein required to inhibit 50% of pyrogallol autooxidation. 2.6.2. Reactive oxygen species (ROS) ROS levels were measured by means of fluorimetric method described by Black [27]. The sample was prepared by homogenizing the heads of 50 flies in 1 ml ice-cold Tris-HCl buffer (0.4 M; pH 7.4) and centrifuged for 10 min with 2000 g at 4 ◦ C. 100 ␮l of filtered supernatant was added to each well of the microplate containing 15 ␮l of 5 ␮M Dichloro-dihydrofluorescein diacetate (DCFH-DA) and the total volume made up to 200 ␮l by adding homogenizing buffer. After incubation at room temperature for an hour, the microplate was read in a spectrofluorometer. The conversion of DCFH-DA to DCF was measured at 489 nm excitation and 525 nm emission wavelengths. 2.6.3. Lipid peroxidation (LPO) Lipid peroxidation assay was carried out following the method of Buege and Aust [28]. To carry out this reaction, 200 ␮l of sample in 5% sucrose solution was added to a reaction mixture containing 200 ␮l of 8.1% SDS, 1.5 ml of 20% acetic acid, 1.5 ml of 0.8% TBA and 0.6 ml of distilled water. The mixture was thoroughly mixed and incubated for 1 h at 95 ◦ C. These samples were cooled under running tap water, to which 3 ml of butanol was added and centrifuged at 8000 g for 5 min. 1 ml of the yellowish supernatant was taken and the absorbance was read at 532 nm. 2.6.4. Glutathione (GSH) The heads of 50 flies were homogenized in ice-cold 10% TCA and 10 mM EDTA (1:1) and centrifuged at 5000 g (15 min at 4 ◦ C). 200 ␮l of the supernatant was added to 3 ml reaction mixture containing

2.6.5. ChE enzymes Acetylcholinesterase (AChE) and Butyrylcholinesterase (BChE) activities were determined by the method of Ellman [30]. To 1 ml of reaction mixture containing sodium-phosphate buffer (0.1 M; pH 8.0), DTNB (10 mM) and 30 ␮l of head homogenate sample, ACTI or BTCI (78 mM) was added. Changes in the absorbance at 412 nm were monitored for 3 min and expressed as nmoles of substrate hydrolyzed/min/mg protein. 2.7. Immunohistochemistry

2.8. Confocal microscopy To visualize the expression and activity of ChAT, the prepared slides were observed under a Zeiss LSM710 confocal microscope (Carl Zeiss, Jena, Germany). In optical sections of the brain obtained by confocal microscopy, the fluorescent signal was visualized at the wavelength of 594 nm. Images were taken at 1 ␮m inter section intervals in 40X with 1024 × 1024 pixel resolution. The confocal images obtained were adjusted to desired contrast and brightness using ZEN 2010 software (Carl Zeiss). 2.9. Real time PCR To investigate age-related changes in the expression levels of sod1 and cat genes, RT-PCR amplification was carried out. Total RNA was extracted from homogenate of heads using TRIzol reagent. The primers were manually designed using Gene Runner version 3.05. The primer sequences were as follows: for sod1 5’-GAACTACTTTGCTGAGGTGG and 3’-GGATCTGCAAGTAGTTCGGT, for catalase 5’-TCAACATCACCGACTCCAAG and 3’-CAGCGTTGCCCGTTGACTT, and for rpl32 5’-AGGGTATCGACAACAGAGTG and 3’-GAACTTCTTGAATCCGGTGG. Primer validation was carried out using control sample and the amplicon sizes were confirmed using Bioanalyzer. Using affinity Script QPCR cDNA synthesis kit, 1 ␮g of DNase treated RNA was reverse transcribed to get 50 ng/␮l of cDNA. Relative quantification was carried out using Brilliant II SYBR Green qPCR Master mix. Each sample was run in duplicate for each gene using 50 ng input per reaction. The experiment was conducted using Stratagene Mx3005P platform. Relative expression level of the genes was determined after normalizing with RPL32 as the reference gene using Delta Ct method [33]. PCR condition was as follows: initial denaturation at 95 ◦ C for 10 min followed by 40 cycles of 95 ◦ C for 30 s, 60 ◦ C for 1 min, 72 ◦ C for 1 min. Melting curve was drawn after completion of the assay to check for the specificity of the reaction.

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3.2. Morphology of Kenyon cells and mushroom bodies (MBs) Light microscopic observation of Hematoxylin-Eosin stained frontal section of the brain from 50-day-old flies revealed higher order of neurodegeneration in cell bodies and neuropile area of Kenyon cells as evident by higher number of vacuolated areas compared with that of 5-day-old flies (Fig. 2). Electron micrographs of MBs of 50-day-old flies showed a reduction in the number of synapses, typical structures showing prominent electron dense strip in pre-synaptic regions surrounded by synaptic vesicles, in a randomly selected 8 ␮m2 area (n = 3). Further, not only the mitochondrial number was reduced, but they were also irregular in size and shape (Fig. 3). Fig. 1. Effect of aging on memory retention. To evaluate age-related memory impairment, STM, MTM and LTM of all the three age groups viz., 5, 25 and 50 days old flies have been examined. Two-way ANOVA revealed a significant decrease in 0 h memory (F (2 , 21) = 61.621, **p < 0.01, n = 8). PI scores of old and mid-aged flies showed significant decrease in 1 h memory (F (2 , 21) = 212.44, ***p < 0.001, n = 8) and 3 h memory (F (2 , 21) = 46.63, ***p < 0.001, n = 8). Old flies showed reduction in long term memory retention at 24 h after 10X spaced training paradigm when compared to the young flies (F (2 , 21) = 32.36, ***p < 0.001, n = 8). There was a significant reduction in anesthesia-resistant memory retention at 24 h after 10X massed training procedure as compared to the young flies (F (2 , 21) = 49.99, ***p < 0.001, n = 8).

2.10. Down and up regulation of antioxidant genes Taking the advantages of UAS-GAL4 system [34], RNAi posttranscriptional gene silencing was achieved by conducting crosses between UAS-cat-IR or UAS-sod-IR lines and MB247 GAL4, as driver line for mushroom bodies. Inverted repeats of cat and sod1 gene sequences are expressed under control of UAS (upstream activating sequence) promoter in the antisense-sense orientation in a particular tissue which is driven by the Gal4 line. The transcribed products form hairpin RNA (hpRNA). These double-stranded RNAs are processed by Diser complex into siRNAs and then direct sequence-specific degradation of the mRNAs corresponding to the target gene [35]. Up regulation of catalase was accomplished by conducting genetic cross between UAS-cat and MB247GAL4 to overexpress cat in MBs.

2.11. Statistical analyses The data were analyzed by t-test and one-way ANOVA followed by ‘Bonferroni’ post-hoc comparisons. The p value of 0.05 was considered as the minimum level of significance.

3. Results 3.1. Effect of aging on memory retention To evaluate age-related memory impairment, we examined the STM, MTM and LTM of all the three age groups viz., 5-day-old (young), 25-day-old (mid age), and 50-day-old (old). We observed a slight but statistically significant decrease in 0 h memory. The PI scores of old and mid-aged flies showed significant decrease in both 1 h and 3 h memories. A significant reduction in LTM retention at 24 h after training and an apparent reduction in anesthesiaresistant memory retention at 30 min after cold shock was observed in old flies compared to the young and mid-aged ones (Fig. 1). Therefore, in old flies there is an obvious decline in consolidated forms of memory. The flies of all genotypes and age groups produced similar distribution scores after exposure to CS+ , CS- and US (Table 1). Such a response clearly indicates the absence of differences in odor perception profile and electric shock response.

3.3. Biochemical and immunohistochemical studies Significant reduction in the activity of the antioxidant enzymes, catalase and SOD, was evident in old flies. Catalase and superoxide dismutase showed significant decrease in their activity by 28.8% and 20%, respectively, in old flies compared to that of young flies. While the levels of ROS and LPO were significantly higher by 24% and 10%, respectively, in the older flies. GSH content further, showed a decrease of 15.65% in old flies compared to the young flies. Our results revealed the absence of significant differences in the activity of the enzymes, AChE and BChE, between different age groups (Fig. 4). Confocal imaging of whole brain samples subjected to ChAT (choline acetyltransferase) antibody staining revealed a significant decrease in the level of this neurotransmitter enzyme in mushroom bodies extrinsic neurons (MBENs) that connect MBs to the antennal lobe projection neurons [36] as well as in lateral horn (LH) neurons that may involve in concentration coding, bilateral processing and multimodal integration of olfactory information [37] in 50-day-old flies (Fig. 5).

3.4. Age-related gene expression of antioxidant enzymes In a quantitative real time PCR, the transcription levels of sod1 and cat genes was compared among different age group flies. Results revealed a significant age-related decrease in the levels of sod1 and cat transcripts (Fig. 6).

3.5. Effect of down and up regulation of antioxidant genes on AMI RNAi-mediated post transcriptional gene silencing of cat and sod1 genes in the MBs of transgenic flies caused a remarkable reduction in 1 h MTM, 3 h MTM and LTM of 10-day old flies compared to that of 3-day-old flies. The memory score of 15-day-old MBRNAi transgenic flies was near to memory retention scores of 50 days old wild type flies. The results indicated that over-expression of cat rescued the flies from deleterious effects of oxidative stress (Fig. 7). Further, 1 h memory performance of 35 days old MB-cat transgenic flies were significantly higher than that of control flies (Fig. 8).

3.6. Decline in memory retention in the long-lived strain We have isolated a long-lived strain (LLS) of D. melanogaster through selective inbreeding [47]. Analysis of 1 h MTM and LTM in the long-lived strain revealed age-related decline in 50-day-old flies. However, the extent of decline in this memory was significantly lower than that of normal lifespan D. melanogaster strain of the same age (Fig. 9).

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Fig. 2. Morphology of Kenyon cells and mushroom bodies. Histology of the Drosophila brain revealed an increased age-dependent neurodegeneration in cell bodies and neuropile area of Kenyon cells. Photomicrographs of the frontal sections of the brain in 5-day-old (a) and 50-day-old (b) flies revealed the vacuolated regions in the neuropile area (arrow heads) and cell bodies (arrows) of Kenyon cells which are accepted indicators for occurrence of apoptosis. Vacuolated areas were measured and analyzed using Image J software and shown as Mean ± SE. The mean size of vacuolated areas in the brain section of young flies and old flies were 280 ␮m2 and 680 ␮m2 , respectively. The brain of 50-day old flies showed significant increase in neurodegeneration (c) (***p < 0.001, by t-test, n = 5).

Fig. 3. Electron microscopy of mushroom bodies. Electron micrographs of a randomly selected 8 ␮m2 area of Drosophila MBs were analyzed. The number of synapses, typical structures showing prominent electron dense strip in pre-synaptic regions surrounded by synaptic vesicles (arrowheads) were counted and compared between 5-day-old (a) and 50-day-old (b) flies. Scale bars represent 2 ␮m. High power electron micrograph in the MBs of 50-day-old flies (c) showed mitochondria with irregular shape and the presence of giant mitochondrial structures. Scale bar represent 500 nm. (d) The mean number of synapses in 8 ␮m2 of 5-day old flies MBs was about 24 while the older flies showed decrease of 29% and the mean number of synapses was about 17 in 50-day old flies (***p < 0.001).

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Table 1 Odor perception and electric shock response. MCHa Age and genotype 5d, OK 5d, MB247/+ 5d, MB; cat-IR 5d, MB; sod-IR 5d, MB; cat 50d, MB; cat-IR 50d, MB;sod-IR 50d, MB; cat 50d, OK 50d, MB247/+

1X 96.3 ± 2.2 96.7 ± 4.4 94.3 ± 1.6 95.3 ± 3.2 96.4 ± 1.4 46 ± 7.5 47 ± 4.1 48 ± 2.2 49 ± 1.5 47.5 ± 1.9

OCTb 0.1X 53.1 ± 2.1 52.7 ± 2.4 51.6 ± 2.5 53.4 ± 2.1 51.8 ± 2.9 26.1 ± 2.7 25.3 ± 1.2 24.8 ± 3.2 26.2 ± 2.8 24.6 ± 2.2

Electric shockc

1X 96.6 ± 1 95.1 ± 1.3 95.2 ± 3.3 94.6 ± 4.1 95.8 ± 2.2 41.2 ± 3.2 40.1 ± 2.7 42.3 ± 2.1 42.6 ± 3.4 40.5 ± 2.3

0.1X 45 ± 3.4 44.7 ± 2.8 46.1 ± 2 45.3 ± 3.5 43.8 ± 2.7 21.4 ± 3.7 22.6 ± 3.1 20.7 ± 2.4 22.1 ± 4.2 22.3 ± 1.9

60V 87.1 ± 3.2 85.5 ±3.6 86.2 ±3.4 84.5 ±3.3 85.8 ±1.7 67.6 ± 3.2 68.1 ± 2.4 67.2 ±3.8 69.4 ±1.3 68.9 ± 2.7

90V 66.3 ± 3.5 66.5 ± 1.1 67.9 ± 3.4 68.2 ± 2.5 67.5 ± 4.1 48.2 ± 3.4 49.5 ± 1.4 48.7 ± 4.1 47.8 ± 3.2 49.3 ± 2.6

a Avoidance of 4-Methylcyclohexanol, b Avoidance of 3-Octanol at the concentration used for conditioning (1X) or a 10-fold dilution (0.1X), n = 6. c Avoidance of electric shock pulses at 60 V or 20 V (n = 6). A two-way ANOVA revealed a significant multivariate main effect for age (Wilk’s ␭ = 0.009, F (6 , 45) = 8.343, P < 0.001) but not for genotype (Wilk’s ␭ = 0.970, F (24 , 158) = 0.57, P = 1.0).

4. Discussion Oxidative stress-induced changes in the cellular organelles and macromolecules in many pathological conditions including neurodegenerative diseases are also associated with the aging process [38]. According to the free radical theory of aging, oxidative stress is implicated in the age-related functional decline [1,39]. Brain is more vulnerable to free radical damages in view of its higher rate of metabolism and lower capacity for regeneration as compared to the other organs [40]. Oxidative stress is considered to be one of the main causal factors involved in the impairment of cognitive function [41,42]. In the current study, we have shown that there is a minor agerelated impairment in STM (0 h memory) but a noticeable reduction in 1 h and 3 h MTM of old flies. Consolidated forms of memory were affected by aging as well. Although old flies showed remarkable decrease in both AMR (anesthesia-resistant memory) and LTM (long-term memory), the latter was more affected. STM is one of

the short-lived memories that is abolished within 30 min after a single training cycle, and relies on functional synaptic trafficking of neurons. There is a possibility that formation of MTM is dependent on STM, occurring downstream of it [43]. ARM formation occurs immediately after continuous training sessions and can last for a few days depending on the intensity of training [19]. Contrary to LTM, inhibition of protein synthesis due to the translationinhibitor, cycloheximide, does not interrupt ARM [18]. Isabel et al. [44] have proposed that ARM exclusively acts as gating mechanism for LTM formation. LTM is a protein-synthesis-dependent phenomenon [18] that critically relies on cAMP-response element binding protein (CREB)-mediated gene expression [45]. Ryu et al. [46] have shown that antioxidant-mediated enhancement of PKA activity results in the increased rate of CREB phosphorylation and thereby amelioration of oxidative stress in neuronal cells. Several studies have reported the accumulation of giant (enlarged) and highly interconnected mitochondrial structures [47,48], low level of ATP production, swollen morphology and loss

Fig. 4. Biochemical assays. Biochemical investigations showed a significant decay in the antioxidant defense system of old flies. Antioxidant enzymes, catalase (a) and superoxide dismutase (b), showed significant decrease in their activity by 28.8% and 20%, respectively, in old flies compared to that of young flies (p < 0.05, by t-test, n = 3). c) GSH showed a decrease of 15.65% in old flies compared to the young flies p < 0.05 by t-test, n = 3). Lipid peroxidation (d) and ROS levels (e) were significantly higher by 24% and 10%, respectively in the old flies (p < 0.05 by t-test, n = 3).

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Fig. 5. Activity of the neurotransmitter enzymes. AchE activity (a) did not show significant changes between the young and old flies (p < 0.8, by t-test, n = 3). BchE activity (b) also did not show significant changes during the aging of flies (p < 0.95, by t-test, n = 3). Photomicrographs showing the activity of ChAT (choline acetyltransferase) in the brain of 5-day-old (c) and 50-day-old (d) flies. The pixel intensity in the mushroom body extrinsic neurons (MBENs) (arrows) and lateral horns (LNs) (arrowheads) areas were estimated using Image J software and relative intensities are shown as Mean ± SE. There were significant decrease in the fluorescent intensity level of ChAT in the selected regions of the brain in old flies compared to the young flies (**p < 0.01) (e). Scale bars represent 50 ␮m.

of cristae in the cells of aging brain [49]. Mitochondria being highly dynamic organelles, can respond and adapt to a variety of intra and extracellular stimuli via change in its morphology and function [39]. Regulated mitochondrial autophagy is crucial for post-mitotic cells possessing low capacity of regeneration [39]. Further, mitochondria are the main sites of free radical generation in the cell [46]. Induction of oxidative stress causes impairment in the cellular homeostasis and defense mechanisms in mt-DNA damages [50] and impaired electron transfer chain [51] that eventually causes cellular aging and death. Age-associated accumulation of oxidative damage in mitochondria can induce imbalanced fission and fusion as well as deregulated mitochondrial autophagy and thereby result in disrupted mitochondrial dynamics [52]. Our biochemical results revealed significant reduction in the activity of the antioxidant enzymes, SOD and catalase, in old flies compared to that of young flies. While the level of ROS and lipid peroxidation were

significantly higher in the old flies, the amount of GSH was lower compared to the young flies. Light microscopic examination of the frontal section of the brain from old flies in the present study revealed higher order of neurodegeneration in cell bodies and neuropile area of Kenyon cells as evident by increased vacuolated areas as compared with that of young flies. Electron micrographs of the MBs from older flies showed a reduction in the number of synapses and presence of giant mitochondria with distorted cristae. In the current study, presence of giant mitochondria in the MBs of old flies could be a consequence of mitochondrial damage induced by the increased cellular oxidative damage due to decay in efficacy of cellular antioxidant defense system. Induction of oxidative stress is also accompanied by reduction in acetylcholine levels in neuronal degeneration [53]. The active brain requires synthesis of acetylcholine as reflected by the activity of choline acetyltransferase (ChAT). Our results showed that due to aging, the activity of the enzyme, ChAT, was decreased whereas

M. Haddadi et al. / Behavioural Brain Research 259 (2014) 60–69

Fig. 6. Gene expression level of cat and sod1. Transcription levels of sod1 and cat were measured in 5 and 50-day-old flies, by quantitative real time PCR. The results revealed a significant decrease in the level of sod1 and cat transcripts due to aging (*p < 0.05, by t-test, n = 3).

the activity of AchE and BchE enzymes was unchanged, a process that could lead to lower level of acetylcholine compromising the neuronal functions in the aged brain. Microarray-based study on age-dependent changes in the expression of genes in Drosophila head has revealed significant reduction in the expression of gene categories involved in oxidative phosphorylation and ubiquitin-dependent proteolytic system [54]. Using real time PCR in present experiment, we have shown that there is a considerable reduction in the expression levels of the antioxidant enzymes, SOD and catalase, in old flies. Moreover, our experiments on RNAi-mediated post transcriptional gene silencing of cat and sod1 genes in the MBs caused a remarkable reduction

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in MTM and LTM while over-expression of cat rescued the AMI in transgenic flies. Previously, we have shown that supplementation of herbal-based antioxidant compounds could attenuate AMI in Drosophila [55] associated with an increase in the total protein content of 55-day-old Drosophila head sample (data not shown). Mery [56] has reported age-dependent decline of LTM but not ARM in Drosophila; based on which it was proposed that natural selection favors formation of ARM form of consolidated memory to balance between costs and benefits especially under extreme stress conditions. In this context, we made a comparative study on consolidated memory using long-lived strain (LLS) of D. melanogaster isolated by Deepashree et al., [57]. Our results revealed the existence of AMI in MTM and LTM in the aged LLS flies. However, the extent of AMI was remarkably lower than normal lifespan strain (NLS) at the same age. Further, biochemical results showed that resistance to oxidative stress is higher in LLS compared to NLS due to enhanced activity of antioxidant enzymes in the LLS flies [58] which favors higher capacity for formation of LTM. In contrast to LTM, ARM is not protein synthesis-dependent and shows lower level of impairment. As there is increased rate of neural cell death and synaptic degeneration in old flies, STM decline could be a consequence of failure in synaptic plasticity. As STM is upstream of MTM, its disruption can lead to impaired MTM. All these findings suggest the involvement of increased oxidative damage and progressive reduction of cellular function, which consequently lead to age-related memory impairment. Andersen [40] proposed that, there would be a cycle of events downstream of oxidative stress that propagate cellular injuries leading to neuronal death. In view of this, it was suggested that antioxidant therapeutic agents could rescue cell death. In our study, we have demonstrated that age-associated accumulation of

Fig. 7. Effect of RNAi-mediated down regulation of antioxidant genes on AMI. Multivariate analysis of variances indicated that, RNAi-mediated post transcriptional gene silencing of the antioxidant genes, cat and sod1 in MBs lead to remarkable age-related reduction in cognitive ability of the transgenic flies compared to the age-related memory impairment values in control flies (F (8 , 120) = 18.456, ***p < 0.001), (n = 8 for each genotype at particular age). Short-term memory (a), 1 h middle-term memory (b), 3 h Middle-term memory (c) and long-term memory (d) of flies have been studied in each genotype at 5th , 25th , and 50th day after their adult eclosion. The 1 h MTM and LTM memory scores of 25-day-old transgenic flies were near to the memory retention scores obtained by wild type 50-day-old flies (1 h MTM: p = 0.131, LTM: p = 0.179, by t-test, n = 8). Bonferroni post-hoc comparisons in MB247/+; cat-IR/+ and MB247/+; sod1-IR/+ lines showed a significant reduction in MTM and LTM PI scores of 25 days old (**p < 0.01) and 55 days old flies (**p < 0.01) compared to MB247/+; + + flies due to antioxidant gene silencing.

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M. Haddadi et al. / Behavioural Brain Research 259 (2014) 60–69

Fig. 8. Effect of over-expression of catalase gene on AMI. Over-expression of cat in the mushroom bodies partially rescued the flies from age-related memory impairment. Two-way ANOVA indicated that, over-expression of cat, rescued the flies from deleterious effect of oxidative stress and memory performance of the transgenic flies were significantly higher than that of control flies. 1 h middle-term memory performance of 25-day-old and 50-day-old MB247/UAS-cat flies were higher than MB247/+; + + flies (F (1 , 42) = 9.851, p < 0.01, n = 8) (a). Long-term memory PIs of 25-day-old and 50-day-old MB247/UAS-cat flies were higher than MB247/+; + + flies (F (1 , 42) = 7.442, **p < 0.01, n = 8) (b). Bonferroni post-hoc comparisons showed a significant increase in PI scores of 25 and 50 days old MB247/UAS-cat flies compared to MB247/+; + + group (**p < 0.01).

References

Fig. 9. Memory retention in long lived strain of Drosophila melanogaster. The agerelated memory impairment in long lived strain was significantly lower than the Drosophila normal lifespan strain. One-way ANOVA analysis have revealed the age-related decline in 1 h MTM of Drosophila long lived strain (a) (F (2 , 21) = 62.7, p < 0.001, n = 8) and age-related decline in LTM of long lived Drosophila strain (b) (F (2 , 21) = 106.16, p < 0.001, n = 8). Comparison between the NLS and LLS indicate that the extent of age-related memory impairment in LLS was significantly lower than the Drosophila NLS (*p < 0.05, by t-test).

oxidative damage and decline in antioxidant defense system lead to impairment in mitochondrial dynamics and induction of neurodegeneration, which, in turn, impair cognitive function. Acknowledgements We thank the Chairman, Department of Studies in Zoology, University of Mysore, Mysore and IOE for the facilities. We also thank the Director, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore for the electron microscopy facility. We also thank Mr. Mohan J. and Mr. Rohith B.N., Department of Studies in Zoology, University of Mysore, for their help.

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