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Chronic choline supplementation improves cognitive and motor performance via modulating oxidative and neurochemical status in rats
Accepted Manuscript Chronic choline supplementation improves cognitive and motor performance via modulating oxidative and neurochemical status in rats
Saiqa Tabassum, Saida Haider, Saara Ahmad, Syeda Madiha, Tahira Parveen PII: DOI: Reference:
S0091-3057(16)30172-1 doi: 10.1016/j.pbb.2017.05.011 PBB 72487
To appear in:
Pharmacology, Biochemistry and Behavior
Received date: Revised date: Accepted date:
17 October 2016 30 March 2017 30 May 2017
Please cite this article as: Saiqa Tabassum, Saida Haider, Saara Ahmad, Syeda Madiha, Tahira Parveen , Chronic choline supplementation improves cognitive and motor performance via modulating oxidative and neurochemical status in rats, Pharmacology, Biochemistry and Behavior (2017), doi: 10.1016/j.pbb.2017.05.011
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TITLE: Chronic Choline Supplementation Improves Cognitive and Motor Performance via Modulating Oxidative and Neurochemical Status in Rats AUTHORS:
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Saiqa Tabassum1, Saida Haider1*,Saara Ahmad2, Syeda Madiha1, Tahira Parveen1,
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AFFILIATION:
Neuropharmacology and Neurochemistry Research Unit, Department of Biochemistry, University of
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Karachi, Karachi-75270, Pakistan1
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Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi 2
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CORRESPONDING AUTHOR: Dr. Saida Haider
University of Karachi, Karachi-75270, Pakistan
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Department of Biochemistry,
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Phone No.: 99261316 EXT: 2577 EMAIL: [email protected]
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ARTICLE TYPE:
Original Research Article CONFLICT OF INTEREST:
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Authors declare that they have no conflict of interest.
ACCEPTED MANUSCRIPT ABSTRACT: Choline, an essential nutrient, accounts for multiple functions in the body and brain. While its beneficial effects on healthy adults are not clear, choline supplementation is important during pregnancy for brain development, in elderly patients for support of cognitive performance and in patients with neurological disorders to reduce memory deficits. Thus, the aim of this study is to investigate whether choline administration in healthy adult rats beneficially impacts cognitive and locomotor performance, and associated oxidative and neurochemical outcomes. Two groups, control and choline, received tap water and choline bitartrate, respectively at the dose equivalent to adequate intake
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for five weeks. Food intake and body weight were monitored daily. Behavioral analysis comprising assessment of
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cognitive performance (by novel object recognition, passive avoidance and Morris water maze test) and locomotor performance (by Open field, Kondziela’s inverted screen and beam walking test) were performed. Following testing,
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rats were decapitated and brain samples collected for estimation of acetylcholine, redox profile and monoamine measurements. The results showed that chronic choline administration significantly improves cognitive and
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locomotor performance accompanied by a reduction in oxidative stress, enhanced cholinergic neurotransmission and monoamine levels in the brain of healthy adult rats. Hence, chronic choline intake was found to improve behavioral, oxidative and neurochemical outcomes in the normal population, so it can be suggested that choline tablets can be
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used as a safe and effective supplement for improving the neurological health of normal individuals and that they might also be beneficial in preventing cognitive and motor disorders later in life.
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KEYWORDS:
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Learning, Memory, Locomotion, Choline Bitartrate, Acetylcholine, Monoamines, Antioxidants
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INTRODUCTION: For a healthy and long life, humans require consumption of a complex set of nutrients (Naber et al. 2015).
Choline is one such vital nutrient, which accounts for multiple functions in the body and brain (Zeisel and da Costa, 2009; Glenn et al. 2012) including cell membrane integrity, methyl group metabolism, cell signaling pathways, lipid transport, myelination, growth factor signaling (Borges et al. 2015), and synthesis of phospholipids, very low density lipoprotein and acetylcholine (ACh) (Zeisel and da Costa, 2009; Glenn et al. 2012; Wallace et al. 2012; Ueland, 2011). According to the US Institute of Medicine’s Food and Nutrition Board, choline is an essential water-
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soluble dietary nutrient (Glenn et al. 2012; Wallace et al. 2012) with an adequate intake (AI) of 550 mg/day for men
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and 425 mg/day for women (Wallace et al. 2012; National Academies Press, 1998). Reports show that choline deficiency is implicated in different metabolic consequences that include fatty liver disease, DNA damage, cell
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apoptosis, altered gene expression and cognitive impairments (Wallace et al. 2012; Ueland, 2011). Previously, it has been reported that average dietary choline intakes among adult men and women were lower than the AI (Wallace et
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al. 2012; Price et al. 2010). In 1998, choline was recognized as ‘an essential nutrient’ (Zeisel and da Costa, 2009), and it was recommended that humans consume choline in their diet for maintenance of normal bodily functions (Glenn et al. 2012; Wallace et al. 2012). Choline supplements are commercially available for treating liver diseases,
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asthma, to prevent neural tube defects during pregnancy (WebMD and Natural Medicines Comprehensive Database, 2009), for clearing fatty liver, maintaining cell membranes, protecting breast tissue and for improving cardiovascular
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function (Powder City). Choline supplements are ineffective for improving memory function and motor activity (WebMD and Natural Medicines Comprehensive Database, 2009). However, studies have shown that following oral
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administration, exogenous choline enters the circulation, crosses the blood–brain barrier (Borges et al. 2015) and can form ACh (Borges et al. 2015; Leermakers et al. 2015). This ACh may be involved in cognitive function (Gandhi et al. 2000), brain development (Ueland, 2011; Leermakers et al. 2015) and may be required for neuronal survival
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(Borges et al. 2015). Researchers have determined that choline supplementation supports brain development (Borges et al. 2015), reduction of neuropathological memory deficits (Wong-Goodrich et al. 2008; Yang et al. 2000),
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cognition (Borges et al. 2015) and hippocampal responsiveness to cholinergic stimulation (Montoya et al. 2000). Evidence that choline content is lower in Alzheimer’s patients suggests that supplementation of choline may improve the cognitive status of dementia patients (Leermakers et al. 2015). Extensive evidence shows memory
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improvement following prenatal choline supplementation (Glenn et al. 2012; Borges et al. 2015; Meck and Williams, 2003). This effect is likely via an increased ACh concentration due to increased choline acetyltransferase activity and decreased acetylcholinesterase activity in the hippocampal cholinergic system (Jadavji et al. 2015). The majority of choline intake studies involve choline supplementation either during pregnancy or in elderly patients, and very few were conducted in adults. Other studies are based on monitoring effects of choline administration in different pathological states including stroke, traumatic brain injury (Borges et al. 2015; Guseva, 2008], dementia (Conant and Schauss, 2004; Lee et al. 2015), and epilepsy (Wong-Goodrich et al. 2008). A recent review (Leermakers et al. 2015) of choline supplementation studies also shows that varying choline intervention durations and use of multiple choline compounds have made clear conclusions regarding the beneficial effects of choline difficult to assess. Effects of choline supplementation specifically in healthy adults is still a matter of debate, as a
ACCEPTED MANUSCRIPT recent study observed improved vasomotor performance and decreased pupil size after choline ingestion (Naber et al. 2015), while others reported no beneficial effects of acute choline supplementation on memory function (Lippelt et al. 2016; Nagrecha et al. 2013). The effects of chronic choline supplementation on cognitive performance and motor function in healthy adult rats have not yet been systematically studied. Therefore, the aim of the current study is to investigate the effects of choline bitartrate tablets on cognitive and locomotor behavior and associated alterations in neurochemical and oxidative systems in healthy adult rats at the dose equivalent to AI recommended in
MATERIALS AND METHODS:
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humans.
2.1. ANIMALS:
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Twelve locally bred Albino-Wistar rats purchased from Dow University of Health Sciences, OJHA campus, Karachi, Pakistan, were used in this study. Animals were caged individually (to avoid effects of social interaction) with ad libitum access to cubes of standard rodent diet and tap water under a 12:12 h light/dark cycle (lights on at
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7:00 am) at controlled room temperature (22±2 °C). For seven days prior to the experiments, animals were subjected to an acclimation period and to various handling procedures to nullify novelty and handling stress. All animal
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experiments were approved by the institutional ethics and animal care committee and performed in strict accordance with the National Institute of Health Guide for Care and Use of laboratory Animals (Publication No. 85-23, revised 1985). All treatments and behavioral monitoring were conducted in a balanced design to avoid order and time
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effects.
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2.2. DRUGS AND CHEMICALS:
Choline bitartrate tablets purchased from Nature’s way, New York, USA were used in the experiment. All chemicals were of analytical grade. All reagents were freshly prepared before the start of the experiment. Drug
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solutions were freshly made in tap water each day for administration at the dose equivalent to AI recommended in humans (500 mg/day) (Wallace et al. 2012). Controls received an equal volume of tap water. Hydrogen peroxide
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(H2O2) stock (35 %) solution, thiobarbituric acid (TBA), trichloroacetic acid (TCA), nitro blue tetrazolium (NBT), and dithiobisnitrobenzoic acid (DTNB) were purchased from the British Drug House (BDH, Dorset, UK). Hydroxylamine hydrochloride (H3NO·HCl), acetylthiocholine (ATC), and all other analytical grade reagents were
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purchased from Sigma Chemical Co. (St. Louis, USA). 2.1. EXPERIMENTAL PROTOCOL: Animals (n=12) (weight, 150-200 g) were randomly divided into two experimental groups (n=6); Control and Choline. Control rats received tap water daily via the oral route in a volume of 0.2 ml/150 g body weight. Test rats received an aqueous solution of choline bitartrate tablet powder via the oral route, at a dose of 52 mg/kg/day body weight daily in a volume of 0.2 ml/150 g body weight, for the duration of five weeks. The dose selection is equivalent to the recommended dose for humans (500 mg/day/60 Kg body weight) mentioned by the manufacturer, and previous reports (Zeisel and da Costa, 2009), and from a pilot study conducted in our laboratory (Tabassum and Haider, 2016). At the end of four weeks’ treatment schedule, behavior studies were performed as outlined in figure
ACCEPTED MANUSCRIPT 1. Behavioral tests included the Open Field test (OFT), the Inverted Screen test (KIST), the Beam Walking test (BWT) to assess locomotor activity, muscular strength and motor coordination, respectively, the Novel Object Recognition test (NORT), Morris Water Maze test (MWM) and the Passive Avoidance test (PAT) to determine recognition ability, spatial and associative learning and memory performance. After monitoring behavioral activities, rats were decapitated, and their brains were removed within 30 s and dissected to collect hippocampal tissue, as
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described previously (Haider et al. 2016).
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Fig. 1: Schematic representation of treatment schedule and Experimental design. 2.2. BEHAVIORAL PROTOCOLS:
Food Intake and Body Weight:
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2.2.1.
Food intake was monitored daily during the five weeks of treatment by giving rats a weighed amount of food
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and weighing the remaining food at next feeding. Body weights of the rats were also monitored daily during the five weeks of the treatment. Animals were weighed at the beginning of the experiment, and were followed up with daily until the end of this study. 2.2.2.
Assessment of Locomotor Activity: Following chronic choline supplementation, OFT, KIST, and BWT were performed to assess locomotor
activity, muscular strength and motor coordination, respectively.
Kondziela's Inverted Screen Test: Kondziela’s inverted screen test has been used previously for measure of muscular strength using all four limbs (Kondziela, 1964). Nearly all healthy animals easily score maximum on this task. The inverted screen is a 43cm square of wire mesh consisting of 12 mm squares of 1 mm diameter wire. It is bordered by a 4 cm deep wooden beading (which prevents animals which attempt to from climbing on
ACCEPTED MANUSCRIPT to the other side). The test was done by placing the rat in the center of wire mesh screen and the screen was rotated to an inverted position over 120 seconds with the rat’s head declining first. The time when the rat falls off from the screen was noted. Animals were scored inverted screen: for 2 min: Falling between 1-10 sec = 1, Falling between 11-25 sec = 2, Falling between 26-60 sec = 3, Falling between 61-90 sec = 4, Falling after 90sec = 5.
Beam Walking Test: Beam walking is a test of motor coordination (Goldstein and Davis, 1990). The rats have to cross a beam which is suspended between a start platform and their home cage at a height of 50 cm and is
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supported by two pillars. A cushion was placed under the beam to protect the animals from the bang into the
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floor. The difficulty of this task can be assorted by using beams with different shapes and widths (Jover et al. 2006). Motor coordination and balance was assessed by the ability of a rat to crossways a graded series of
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beams. Three circular beams of different diameter were used in this study such as 3 cm, 2 cm, 1 cm and length of 100 cm. In the training phase animals were trained to traverse the beam (from widest to narrowest) directly
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into the animal's home cage. This helps to make certain that the behavior during testing is more stable and more precisely reflects motor coordination as opposed to the rodent’s natural aversion to crossing over unprotected spaces. After training session testing phase was done, the time taken to cross the beam and number of foot slips
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off the beam was determined. The foot slips off the beam made by rat was scored on a scale of 0-6 as follows: 0 points, the rat was not able to stay on the beam, 1 point; the rat did not move but able to stay on beam, 2 points;
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the rat tried to traverse beam but fell, 3points; the rat traversed the beam with multiple slips (4-6), 4 points; the rat traversed the beam with few foot slips (2 or 3), 5 points; the rat traversed the beam with only one slip of the
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hind limb, 6 points; the rat traversed the beam without any slip of hind limb (Puurunen, 2001). Assessment of Memory Performance:
The memory performance of rats following choline supplementation was determined across three domains i.e.,
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recognition, spatial and associative memory function by using NORT, MWM and PA tests, respectively. The details of apparatus used and procedures followed was essentially the same as described by Haider et al. (2016).
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2.3. OXIDATIVE AND NEUROCHEMICAL ANALYSIS: Oxidative status was analyzed according to Haider et al. (2015; 2016) to estimate lipid peroxidation (LPO),
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catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities, and GSH and protein content. ACh content and AChE activity was estimated by the same method as described by Batool et al. (2016) and Haider et al. (2015), respectively. Analysis of monoamines and their major metabolites [norepinephrine (NA), dihydroxyphenyl acetic acid (DOPAC), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), and 5 hydroxytryptamine (5-HT)] was performed in rest of the brain and the hippocampus using high-performance liquid chromatography with electrochemical detection (HPLC-EC), in the same manner as described by Haider et al. (2016). 2.4. STATISTICAL ANALYSIS:
ACCEPTED MANUSCRIPT All data are presented as the mean ± S.D. Statistical analyses were performed using SPSS software version 20.0 for windows. All the parameters were tested with the Shapiro-Wilks test and found to be normally distributed (p<0.05); therefore, in instances of multiple mean comparisons, the results of the behavioral and neurochemical assessment were analyzed by Student’s t-test for the two experimental groups for each parameter. Escape latencies during acquisition trials in MWM were analyzed by two-way ANOVA (repeated measures) followed by multiple comparisons by Bonferroni’s test. The level of significance for all comparisons was set at p≤0.05. 3.
RESULTS:
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The objective of this study is to investigate the effects of chronic choline supplementation in healthy adult
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rats on locomotor performance and learning and memory functions, along with determining changes in associated oxidative and neurochemical profiles in brain and hippocampus. These data may support future use of choline as a
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dietary supplement for improving locomotion and cognitive function. Food intake and body weight were not affected following all treatments as shown in table 1.
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TABLE 1: The effects of chronic choline intake on daily food intake and the body weight of animals before and after treatment. Data are presented as the mean ± SD (n = 6). A non-significant effect was obtained by the
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independent sample t-test when compared with control rats as well as with pretreatment values. FOOD INTAKE (g)
SALINE
CHOLINE
10.92±0.54
162.17±1.71
158.24±1.45
10.96±0.44
165.55±1.86
161.25±1.29
CHOLINE
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SALINE
BODY WEIGHT (g)
10.97±0.35
WEEK 2
10.97±0.41
WEEK 3
10.26±1.08
9.89±0.19
166.23±1.34
162.12±1.38
WEEK 4
10.33±0.34
10.21±1.72
165.63±0.92
160.14±0.97
WEEK 5
10.11±0.64
10.01±0.33
165.55±0.75
160.06±0.86
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WEEK 1
Effect of choline on the locomotor function of rats was evaluated via assessing locomotor activity,
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muscular strength and motor coordination by using OFT, KIST and BWT. The results showed that chronic choline supplementation improved the locomotor performance of adult rats. The results of OFT revealed significant
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improvement in locomotor activity evident by a significant reduction in movement latency (t = 4.092, p<0.01) from central area (fig. 2a), and a significant increase in square crossings in choline supplemented adult rats during five min time span, compared to control rats (t = -3.393, p<0.01; fig. 2b).
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Fig. 2: The effect of chronic choline administration on locomotor activity was evaluated by the open field test in terms of movement latency from the central square (A), and the number of squares crossed (B). For each group
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n=6; values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are
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expressed as *=p<0.05, **=p<0.01, compared to control. An analysis of the data obtained during KIST showed that following chronic choline supplementation,
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muscular strength of adult rats was improved, which was evident by a significant increase in fall latencies from
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the inverted screen in choline treated group compared to control group (t = -9.899, p<0.01; fig. 3).
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Fig. 3: The effect of chronic choline administration on muscular strength was evaluated by Kondzeila’s inverted screen test in terms of latency to fall from screen, which were then scored as reported in materials and methods. For each group, n=6; values are presented as the mean ± S.D. Significant differences were obtained
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by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control. Motor coordination assessed by BWT was also improved following chronic choline supplementation indicated by a significant reduction in latency to cross the beam (fig. 4) across three different diameters include 3 cm beam (t = 8.838, p<0.01), 2 cm beam (t = 8.598, p<0.01) and 1 cm beam (t = 7.727, p<0.01); no significant change was observed in scores for foot slips made during beam crossings.
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Fig. 4: The effect of chronic choline administration on motor coordination was evaluated by Beam walking
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test in terms of latency (sec) to cross the beam (A) of 3 different diameters (3 cm, 2 cm and 1 cm) without foot slips (B) while crossing the beam, which were then scored as reported in materials and methods. For each
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group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-
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test and are expressed as *=p<0.05, **=p<0.01, compared to control. The effects of chronic choline supplementation on learning and memory performance and cognitive ability was evaluated by NORT, MWM and PAT that are used to assess recognition, spatial reference and
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associative learning and memory performance, respectively. Recognition memory was assessed by ORT in terms of % exploration and discrimination index presented in fig. 5 (a and b). It was observed that recognition memory was improved in the choline treated group, evidenced by a significant decline in % exploration (fig. 5a) for familiar objects (t = 35.11, p<0.01) and a significant increase in % exploration for novel objects (t = 35.11,
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p<0.01), along with a significant increase in discrimination index (fig. 5b) (t = 39.338, p<0.01) in the choline
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treated group compared to the control group.
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Fig. 5: The effect of chronic choline administration on recognition memory was evaluated by the Novel object recognition task in terms of % exploration (A), and discrimination index (B). For each group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control.
ACCEPTED MANUSCRIPT The effects on spatial cognitive abilities of animals were determined using the MWM task. To find the difference in escape latencies of animals during four acquisition trials, two-way ANOVA (repeated measures) was performed. Our results indicated significant effect of trials (F(4,140)=847.142, p<0.01), groups (F(6,35)=50.465, p<0.01) and interaction between trials x groups (F(24,140)=16.787, p<0.01). Pair-wise comparisons by the Bonferroni test revealed that escape latencies of animals decreased significantly (p<0.01) over trials (shown in figure 6a), which may be due to familiarization to the environment and due to adaptation to the repeated training trials. Analysis of cumulative escape latencies of all acquisition trials revealed a significant decline (t = 6.676, p<0.01) in choline treated rats compared to control rats (figure 6b). Reference memory was
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examined by performing probe trials (free swimming without platform) in MWM tank after 1 hr. and 24 hr. of the last training trial, via monitoring the time spent in the target quadrant (NW) and the number of entries over
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the target quadrant, time taken to reach the target quadrant (quadrant latency), and time taken to reach the platform location (platform latency). Data analysis by Student’s t-test revealed significant increases in time
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spent in the target quadrant (t = 4.681, p<0.01), the number of entries over the target quadrant (t = 3.862, p<0.01), quadrant latency (t = 8.981, p<0.01) and platform latency (t = 8.019, p<0.01) during the 1 hr. probe trial (figure 6c) in the choline supplemented group compared to control group. Significant increases in time
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spent in the target quadrant (t = 5.314, p<0.01), number of entries over target quadrant (t = 6.01, p<0.01), quadrant latency (t = 7.274, p<0.01) and platform latency (t = 9.509, p<0.01) during 24 hr. probe trial (figure 6d) were observed in the choline supplemented group, compared to the control group. These results suggest that
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spatial reference memory acquisition and retention improved following chronic choline supplementation. b)
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Fig. 6: The effect of chronic choline administration on spatial memory performance of rats was determined in
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a spatial memory task [Morris Water Maze (MWM)]. (A) Escape latencies (sec) of rats in a spatial memory task during exposure to acquisition training trials, which were measured for 120 seconds for each trial. For each group n=6; values are presented as the mean ± S.D. (B) Averaged escape latencies (sec) for each animal in
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both groups are analyzed by Student’s t-test, and significant differences are presented as *=p<0.05, **=p<0.01, compared to control. During the probe trials performed after 1 hr. (C) and 24 hr. (D) after last acquisition trial, spatial memory performance was evaluated by monitoring duration of time spent (sec) in the
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target quadrant (NW) in absence of the platform, the number of entries made by each rat over the target quadrant (NW), time (sec) to enter into the target quadrant (Quadrant latency) and time (sec) to reach the platform location (platform latency) during duration of 120 seconds when the platform was removed. For each
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group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Students’ t-
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test and are expressed as *=p<0.05, **=p<0.01, compared to control. Associative memory was assessed by PAT in terms of the difference between pre-training and posttraining step-through latencies to enter the dark compartment in both acquisition and retention phases (fig. 7).
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Associative memory improved following treatment with choline, as evidenced by significant increases in the difference in step-through latencies during acquisition (t = 11.475, p<0.01) and retention phases (t = 40.277, p<0.01). Data analysis of step-through latencies evaluated during the acquisition phase by Student’s t-test
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revealed significant increases (t = 12.33, p<0.01) in step-through latencies of choline supplemented rats compared to control group as shown in figure 7. Similarly, Student’s t-test analysis for step-through latency evaluated during retention also showed significant increases (t = 26.19, p<0.01) in step-through latencies of choline supplemented rats compared to control group, as shown in figure 7.
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Fig. 7: The effect of chronic choline administration on avoidance memory performance of rats was assessed in
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the Passive avoidance test (PA) for 5 min by recording the step-through latencies (sec) during the initial training trial, during the acquisition phase 60 minutes after the initial training trial, and during the retention
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phase, 24 hr. after the initial training trial. The difference of step-trough latencies of the test trial (the time taken by rats to enter the aversive stimulus-associated dark compartment) to step-trough latency of initial training trial (the time required for rats to enter into the dark compartment) was computed for each animal as
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an index of memory deficit. For each group n=6; values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control.
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The effects of chronic choline supplementation on the brain oxidative profile were determined in terms of lipid peroxidation levels (MDA), antioxidant enzyme activities (CAT, GPX and SOD) and levels of antioxidant compounds (GSH and protein) in the brain, are shown in figure 8. The results show that following
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chronic choline supplementation, there was a significant decline in lipid peroxidation levels (MDA) (t = 6.348, p<0.01; fig. 8a), a marked increase in antioxidant enzymes (fig. 8b); GPx (t = 11.422, p<0.01), CAT (t = 12.248,
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p<0.01) and SOD (t = 5.477, p<0.01). There was also a significant increase in levels of antioxidant compounds (fig. 8c); GSH (t = 6.612, p<0.01) and protein content (t = 6.414, p<0.01) in brains of choline treated rats
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compared to controls.
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Fig. 8: The effect of chronic choline administration on the oxidative profile of rats was assessed via
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determination of the levels of lipid peroxidation (A), levels of antioxidant [GSH(nmol/g) and Protein(g/g)] (B) and levels of antioxidant enzymes [CAT (µmol/min/g), GPX (µmol/min/g), SOD (U/g)] (C). For each group,
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n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control.
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Levels of Acetylcholine (ACh) and its metabolizing enzyme acetylcholine esterase (AChE) in the brain were determined by spectrophotometric analysis. ACh levels were significantly increased (t = 5.77, p<0.01) in choline treated rats compared to the control group, as shown in figure 9a. AChE activity significantly declined (t
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= 7.574, p<0.01) in choline treated rats compared to the control group, as shown in figure 9b. These results suggest that brain ACh content was increased following intake of choline via reduced AChE activity. b)
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Fig. 9: The effect of chronic choline administration on the cholinergic profile of rats was assessed via determination of acetylcholine content (A) and ACHE activity (B). For each group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control. The levels of monoamines (NA, DA and 5-HT) and their metabolites (DOPAC, HVA and 5-HIAA) were estimated using HPLC-EC in rest of the brain and hippocampal samples of both groups. Monoamine levels increased following choline treatment. An analysis of the NA levels showed a marked increase in rest of brain (t = 3.48, p<0.01) and hippocampus (t = 5.148, p<0.01) of choline-treated rats compared to control rats (figure 10a). Data analysis on dopamine and its metabolites; DOPAC and HVA by Student’s t-test revealed significant increase in DA levels in both in rest of brain (t = 2.23, p<0.05) and hippocampus (t = 6.957, p<0.01),
ACCEPTED MANUSCRIPT while its metabolite DOPAC was significantly increased in rest of brain (t = 4.33, p<0.01) and decreased in hippocampus (t = 4.696, p<0.01) while no significant change was observed in HVA levels in both rest of brain and hippocampus (figure 10b). Analysis of levels of 5-HT and its metabolite 5-HIAA showed that choline supplementation altered serotonin metabolism as there was a significant increase in 5-HT levels in both rest of brain (t = 10.081, p<0.01) and hippocampus (t = 8.249, p<0.01) and 5-HIAA levels were increased in rest of brain (t = 5.678, p<0.01) while decreased in hippocampus (t = 5.737, p<0.01) (figure 10c). These results suggest that following chronic choline supplementation neurochemical profile was improved as there was a marked increase in monoamine metabolism.
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Fig. 10: The effects of chronic choline administration on the monoamine profile of rats in rest of brain and hippocampus samples were assessed via determining the levels of NA (A), DA and its metabolites (DOPAC & HVA)
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(B), levels of 5-HT and its metabolite 5-HIAA (C). For each group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01,
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compared to control. DISCUSSION:
The aim of current study is to reveal that whether chronic choline supplementation could exert a beneficial
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effect on cognitive function and locomotor performance of healthy adult rats receiving choline at a dose equivalent to AI for the period of five weeks. Our findings show that chronic choline intake is beneficial in improving memory
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function and locomotor performance in healthy adult rats at an appropriate dose, in contrast to previous reports that observed no benefit of choline supplementation on memory function in healthy individuals (Lippelt et al. 2016) and
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in rats, but was only effective when administered in combination with caffeine (Nagrecha et al. 2013). However, our findings are consistent with an earlier study that reported beneficial effects of acute choline supplementation on
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motor performance (Naber et al. 2015). However, the effects on memory function in healthy adult rats and associated oxidative and neurochemical alterations were not addressed. Thus, our study may be the first to assess the beneficial effects of chronic choline supplementation on both memory function and locomotor performance in
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healthy young rats at a dose equivalent to AI. Studies show that dietary choline supplementation during development produces life-long modifications in the brain and subsequent improved behavioral performance, including structural, neurochemical, and electrophysiological alterations responsible for enhancement in cognitive functioning (Glenn et al. 2012; Meck and Williams, 2003; Schneider and Thomas, 2016). Moreover, as the precursor of acetylcholine, choline is also reported to improve cognitive performance in elderly patients (Leermakers et al. 2015). However, in healthy populations, the favorable effects of choline intake have not been observed. Differences in dosage and type of choline used may account for this (Leermakers et al. 2015). Hence, the question may be raised as to whether the effect of choline supplementation is dependent on age and health status. Thus, our study monitored the effects of choline intake in healthy adult rats rather than in diseased or aged rats. Previously we reported that memory function was improved
ACCEPTED MANUSCRIPT following chronic choline intake, via modulation of acetylcholine levels in the brain (Tabassum and Haider, 2016). The present study was designed to investigate the effectiveness of chronic choline supplementation in improving memory and locomotor performance, and in the modulation of oxidative and neurochemical status of a healthy brain. Consistent with our previous study, present findings show that along with inducing changes in behavioral performance, chronic choline intake at AI also has a positive effect on the oxidative and neurochemical status of the brain, indicating that dietary choline administration is beneficial for improving the physical and mental health of adult rats.
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In previous reports, choline is typically administered in diet or water (Borges et al. 2015; Guseva, 2008),
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but few studies administered choline orally (Borges et al. 2015; Wallace et al. 2012). Furthermore, very few studies used a choline dose that was equivalent to AI (Wallace et al. 2012). However, it is evident that the effects of choline
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are dependent upon its dosage (Borges et al. 2015), as smaller doses are not neuroprotective and high doses produce toxic effects such as hypo-activity, hypothermia, hypotension, cyanosis, and high mortality, while moderate doses
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are beneficial (Borges et al. 2015). Commercially available choline tablets (recommended for enhanced absorption and for treatment of liver disease) were used at a dose equivalent to AI (Wallace et al. 2012; National Academies Press, 1998) in our study. Previously, studies of oral choline administration in adults have administered either a
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single dose (Naber et al. 2015) or doses over seven days (Borges et al. 2015), while we orally administered choline daily for five weeks. Food intake and body weight of rats were monitored throughout the experiment. Changes in
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food intake and body weight of rats following choline supplementation were not significantly different than figures reported in previous studies (Leermakers et al. 2015; Killgore, 2010). Previously, some researchers have observed
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adverse effects following choline administration, so we monitored animals throughout the experiment for such effects as mortality and hypo-activity, but no adverse effects were noted in our study (Borges et al. 2015; Unal et al. 1998). Thus, these data suggest that choline administration at doses equivalent to AI is probably safe.
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Because choline is a component of both cell membranes and neurotransmitters involved in nerve signaling, it has a unique role in memory preservation and prevention of cognitive decline (Wood and Allison, 1982).
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Numerous studies report enhancement in rodents’ memory performance following choline intake (Naber et al. 2015; Leermakers et al. 2015). However, studies show positive effects on cognitive status with choline supplementation during pregnancy (Glenn et al. 2012; Borges et al. 2015; Meck and Williams, 2003) in elderly patients (Leermakers
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et al. 2015), or when given in combination with other compounds such as caffeine (Nagrecha et al. 2013), or when given during/after pathological conditions such as dementia (Naber et al. 2015; Blake et al. 2012), ischemia (Borges et al. 2015), traumatic brain injury (Borges et al. 2015; Guseva et al. 2008) or alcohol exposure (Schneider and Thomas, 2016) in rodents or in postmenopausal women (Wallace et al. 2012) but very few studies assessed choline effectiveness when given alone to healthy adults. A recent review reported that choline was not significantly beneficial in improving cognition in healthy patients (Leermakers et al. 2015); while a similar study also reported that choline was not effective when given alone in healthy subjects (Lippelt et al. 2016; Nagrecha et al. 2013). In contrast, our findings suggest that chronic choline administration is effective in improving memory performance of healthy adult rats. Our results showed that choline administration not only improved spatial reference memory but also improved recognition and associative memory performance in rats. This improvement might be because choline
ACCEPTED MANUSCRIPT is the precursor of ACh (Leermakers et al. 2015), so choline administration increases ACh synthesis and release in cholinergic neurons (Sarter and Parikh, 2005), which may be beneficial for neurological health of adult rats, or this might be due to increased availability of phosphatidylcholine that positively affects the neuronal membranes and in turn improves cognitive function (Leermakers et al. 2015; Conant and Schauss, 2004). Along with assessing cognitive function, we also assessed the locomotor performance of rats after choline intake because studies have shown that, besides cognition, cholinergic brain networks are also involved in motor functions (Naber et al. 2015; Woolf, 1991). Our findings show that locomotor activity, muscular strength and motor
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coordination, assessed by OFT, KIST, and BWT, were significantly improved following chronic choline
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administration. This improvement in locomotor performance was consistent with previous studies, which suggest a crucial role of choline in motor functions (Naber et al. 2015). Earlier studies reported enhancement in locomotor
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activity following an increase in choline release in hippocampus and cortex (Toide, 1989). Impaired motor coordination and motor learning after dietary choline deprivation (Pacelli et al. 2010), and attenuation of motor
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deficits by postnatal choline supplementation have also been shown earlier (Arazi et al. 2014). The probable mechanism responsible for improved locomotor performance is increased ACh synthesis in the brain following choline administration. It is reported that in the brain, choline-to-acetylcholine synthesis has an important role
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(Naber et al. 2015) that may affect the cholinergic motor neurons via increasing calcium release from sarcoplasmic reticulum, leading to muscle contraction (Lowes et al. 2013), and may increase motor coordination and locomotor
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activity.
In addition to observing behavioral changes following choline administration, the present study has also
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determined the effect of chronic choline administration on alterations in brain redox status and neurochemical changes in brain and hippocampus. Choline is part of the mitochondrial membrane, the main source of intracellular reactive oxygen species (ROS) and is required for cellular signaling. However, ROS can also cause cell damage, so
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a firm control by endogenous antioxidant scavenging systems exists that limits this damage (Lowes et al. 2013). These are comprised of antioxidant enzymes, including SOD, which catalyzes the dismutation of superoxide to
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H2O2, and CAT or GPX, which then decomposes H 2O2 to water (Dumont et al. 2009), and non-enzymatic antioxidants, such as GSH and protective proteins (Halliwell, 2011). Previously, it has been determined that oxidative stress was enhanced following dietary choline deprivation. This effect is mediated by increased ROS
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production and lipid peroxidation (increased MDA levels), decrease content of GSH and proteins, and reduced activities of SOD and CAT (Santos et al. 2015). The findings in the present paper showed that chronic choline administration reduces the oxidative stress as it reduces the production of ROS via decreasing the levels of lipid peroxidation, stimulating the activities of antioxidant enzymes (CAT, GPX and SOD) and by increasing the antioxidant stores of GSH and proteins. These results are consistent with the studies that reported a reduction in oxidative stress following choline administration in various pathological conditions (Mehta et al. 2010) via decreasing lipid peroxidation. It is evident that administered choline is incorporated into ACh, so by relating choline administration with central cholinergic activity, we found that brain acetylcholine concentrations increased following choline administration, consistent with previous reports showing declines in ACh content following dietary choline
ACCEPTED MANUSCRIPT deficiency (Wecker, 1979); but contrast with studies reporting that choline supplementation modulates ACh synthesis only in certain neurological disorders or after drug-induced neuronal activity, but under normal conditions remains unaltered (Wecker, 1986). In the present study, neurochemical alterations following chronic choline administration were also investigated. We observed that choline administration positively affected the activity of catecholaminergic and serotonergic neurons both in the whole brain and in hippocampus, as there was a marked rise in levels of noradrenaline, modulation of dopamine and serotonin metabolism in brain and hippocampus via increasing their synthesis and release in respective neurons. These alterations might occur via affecting the
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catecholaminergic neurons and by increasing the activity of tyrosine hydroxylase, as cholinergic drugs are known to
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enhance both dopamine synthesis and turnover (Ulus and Wurtman, 1976). However, in contrast, some studies report that choline administration did not alter neurochemical profiles (Wecker, 1979). Thus, our findings suggest
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that as the precursor of ACh, choline not only affects cholinergic neurotransmission but also modulates catecholaminergic and serotonergic neurotransmission leading to improvement in motor performance and memory
5.
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function. CONCLUSION:
The findings of the present study, together with previous findings, suggest that choline administration is
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beneficial for neurological health of not only children or elderly patients but also healthy adults, in particular improving cognitive and locomotor performance. In addition, chronic choline intake was found to improve
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behavioral, oxidative and neurochemical outcomes in the normal population. Although choline supplements are commercially available, these are considered ineffective in improving brain function, particularly memory and
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motor performance. Thus, our current findings suggest that chronic choline administration at doses equivalent to AI might have a positive impact on brain function, particularly on cognitive and motor function in healthy adult rats. These findings suggest the use of available choline supplements for the neurological functioning in humans may be
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beneficial. Thus, it may be suggested that in the future choline may be used as a safe and effective supplement for improving the neurological health status of normal individuals and that it might also be beneficial in preventing
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cognitive and motor disorders later in life. ACKNOWLEDGEMENTS:
English. 7.
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Authors are grateful to Prof. Hafiz Syed Ali Athar for critically going through the manuscript for improving the
FUNDING AND DISCLOSURE: The funds for experimental material and animals for the present study were provided by Higher Education
Commission (HEC), Pakistan and University of Karachi, Karachi, Pakistan. Authors have no competing financial interests in relation to the work described. 8.
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Graphical abstract
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HIGHLIGHTS Study was aimed to investigate whether choline intake is beneficial in healthy adults.
Chronic choline intake at an adequate dose improved brain functioning in healthy adults
Chronic choline administration improved cognitive and locomotor function.
Choline reduced oxidative stress, enhanced cholinergic and monoaminergic transmission.
Hence, choline tablets may be suggested as a safer and effective supplement to improve neurological
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health.
Saiqa Tabassum, Saida Haider, Saara Ahmad, Syeda Madiha, Tahira Parveen PII: DOI: Reference:
S0091-3057(16)30172-1 doi: 10.1016/j.pbb.2017.05.011 PBB 72487
To appear in:
Pharmacology, Biochemistry and Behavior
Received date: Revised date: Accepted date:
17 October 2016 30 March 2017 30 May 2017
Please cite this article as: Saiqa Tabassum, Saida Haider, Saara Ahmad, Syeda Madiha, Tahira Parveen , Chronic choline supplementation improves cognitive and motor performance via modulating oxidative and neurochemical status in rats, Pharmacology, Biochemistry and Behavior (2017), doi: 10.1016/j.pbb.2017.05.011
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TITLE: Chronic Choline Supplementation Improves Cognitive and Motor Performance via Modulating Oxidative and Neurochemical Status in Rats AUTHORS:
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Saiqa Tabassum1, Saida Haider1*,Saara Ahmad2, Syeda Madiha1, Tahira Parveen1,
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AFFILIATION:
Neuropharmacology and Neurochemistry Research Unit, Department of Biochemistry, University of
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Karachi, Karachi-75270, Pakistan1
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Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi 2
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CORRESPONDING AUTHOR: Dr. Saida Haider
University of Karachi, Karachi-75270, Pakistan
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Department of Biochemistry,
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Phone No.: 99261316 EXT: 2577 EMAIL: [email protected]
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ARTICLE TYPE:
Original Research Article CONFLICT OF INTEREST:
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Authors declare that they have no conflict of interest.
ACCEPTED MANUSCRIPT ABSTRACT: Choline, an essential nutrient, accounts for multiple functions in the body and brain. While its beneficial effects on healthy adults are not clear, choline supplementation is important during pregnancy for brain development, in elderly patients for support of cognitive performance and in patients with neurological disorders to reduce memory deficits. Thus, the aim of this study is to investigate whether choline administration in healthy adult rats beneficially impacts cognitive and locomotor performance, and associated oxidative and neurochemical outcomes. Two groups, control and choline, received tap water and choline bitartrate, respectively at the dose equivalent to adequate intake
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for five weeks. Food intake and body weight were monitored daily. Behavioral analysis comprising assessment of
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cognitive performance (by novel object recognition, passive avoidance and Morris water maze test) and locomotor performance (by Open field, Kondziela’s inverted screen and beam walking test) were performed. Following testing,
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rats were decapitated and brain samples collected for estimation of acetylcholine, redox profile and monoamine measurements. The results showed that chronic choline administration significantly improves cognitive and
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locomotor performance accompanied by a reduction in oxidative stress, enhanced cholinergic neurotransmission and monoamine levels in the brain of healthy adult rats. Hence, chronic choline intake was found to improve behavioral, oxidative and neurochemical outcomes in the normal population, so it can be suggested that choline tablets can be
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used as a safe and effective supplement for improving the neurological health of normal individuals and that they might also be beneficial in preventing cognitive and motor disorders later in life.
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KEYWORDS:
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Learning, Memory, Locomotion, Choline Bitartrate, Acetylcholine, Monoamines, Antioxidants
ACCEPTED MANUSCRIPT 1.
INTRODUCTION: For a healthy and long life, humans require consumption of a complex set of nutrients (Naber et al. 2015).
Choline is one such vital nutrient, which accounts for multiple functions in the body and brain (Zeisel and da Costa, 2009; Glenn et al. 2012) including cell membrane integrity, methyl group metabolism, cell signaling pathways, lipid transport, myelination, growth factor signaling (Borges et al. 2015), and synthesis of phospholipids, very low density lipoprotein and acetylcholine (ACh) (Zeisel and da Costa, 2009; Glenn et al. 2012; Wallace et al. 2012; Ueland, 2011). According to the US Institute of Medicine’s Food and Nutrition Board, choline is an essential water-
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soluble dietary nutrient (Glenn et al. 2012; Wallace et al. 2012) with an adequate intake (AI) of 550 mg/day for men
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and 425 mg/day for women (Wallace et al. 2012; National Academies Press, 1998). Reports show that choline deficiency is implicated in different metabolic consequences that include fatty liver disease, DNA damage, cell
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apoptosis, altered gene expression and cognitive impairments (Wallace et al. 2012; Ueland, 2011). Previously, it has been reported that average dietary choline intakes among adult men and women were lower than the AI (Wallace et
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al. 2012; Price et al. 2010). In 1998, choline was recognized as ‘an essential nutrient’ (Zeisel and da Costa, 2009), and it was recommended that humans consume choline in their diet for maintenance of normal bodily functions (Glenn et al. 2012; Wallace et al. 2012). Choline supplements are commercially available for treating liver diseases,
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asthma, to prevent neural tube defects during pregnancy (WebMD and Natural Medicines Comprehensive Database, 2009), for clearing fatty liver, maintaining cell membranes, protecting breast tissue and for improving cardiovascular
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function (Powder City). Choline supplements are ineffective for improving memory function and motor activity (WebMD and Natural Medicines Comprehensive Database, 2009). However, studies have shown that following oral
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administration, exogenous choline enters the circulation, crosses the blood–brain barrier (Borges et al. 2015) and can form ACh (Borges et al. 2015; Leermakers et al. 2015). This ACh may be involved in cognitive function (Gandhi et al. 2000), brain development (Ueland, 2011; Leermakers et al. 2015) and may be required for neuronal survival
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(Borges et al. 2015). Researchers have determined that choline supplementation supports brain development (Borges et al. 2015), reduction of neuropathological memory deficits (Wong-Goodrich et al. 2008; Yang et al. 2000),
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cognition (Borges et al. 2015) and hippocampal responsiveness to cholinergic stimulation (Montoya et al. 2000). Evidence that choline content is lower in Alzheimer’s patients suggests that supplementation of choline may improve the cognitive status of dementia patients (Leermakers et al. 2015). Extensive evidence shows memory
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improvement following prenatal choline supplementation (Glenn et al. 2012; Borges et al. 2015; Meck and Williams, 2003). This effect is likely via an increased ACh concentration due to increased choline acetyltransferase activity and decreased acetylcholinesterase activity in the hippocampal cholinergic system (Jadavji et al. 2015). The majority of choline intake studies involve choline supplementation either during pregnancy or in elderly patients, and very few were conducted in adults. Other studies are based on monitoring effects of choline administration in different pathological states including stroke, traumatic brain injury (Borges et al. 2015; Guseva, 2008], dementia (Conant and Schauss, 2004; Lee et al. 2015), and epilepsy (Wong-Goodrich et al. 2008). A recent review (Leermakers et al. 2015) of choline supplementation studies also shows that varying choline intervention durations and use of multiple choline compounds have made clear conclusions regarding the beneficial effects of choline difficult to assess. Effects of choline supplementation specifically in healthy adults is still a matter of debate, as a
ACCEPTED MANUSCRIPT recent study observed improved vasomotor performance and decreased pupil size after choline ingestion (Naber et al. 2015), while others reported no beneficial effects of acute choline supplementation on memory function (Lippelt et al. 2016; Nagrecha et al. 2013). The effects of chronic choline supplementation on cognitive performance and motor function in healthy adult rats have not yet been systematically studied. Therefore, the aim of the current study is to investigate the effects of choline bitartrate tablets on cognitive and locomotor behavior and associated alterations in neurochemical and oxidative systems in healthy adult rats at the dose equivalent to AI recommended in
MATERIALS AND METHODS:
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humans.
2.1. ANIMALS:
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Twelve locally bred Albino-Wistar rats purchased from Dow University of Health Sciences, OJHA campus, Karachi, Pakistan, were used in this study. Animals were caged individually (to avoid effects of social interaction) with ad libitum access to cubes of standard rodent diet and tap water under a 12:12 h light/dark cycle (lights on at
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7:00 am) at controlled room temperature (22±2 °C). For seven days prior to the experiments, animals were subjected to an acclimation period and to various handling procedures to nullify novelty and handling stress. All animal
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experiments were approved by the institutional ethics and animal care committee and performed in strict accordance with the National Institute of Health Guide for Care and Use of laboratory Animals (Publication No. 85-23, revised 1985). All treatments and behavioral monitoring were conducted in a balanced design to avoid order and time
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effects.
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2.2. DRUGS AND CHEMICALS:
Choline bitartrate tablets purchased from Nature’s way, New York, USA were used in the experiment. All chemicals were of analytical grade. All reagents were freshly prepared before the start of the experiment. Drug
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solutions were freshly made in tap water each day for administration at the dose equivalent to AI recommended in humans (500 mg/day) (Wallace et al. 2012). Controls received an equal volume of tap water. Hydrogen peroxide
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(H2O2) stock (35 %) solution, thiobarbituric acid (TBA), trichloroacetic acid (TCA), nitro blue tetrazolium (NBT), and dithiobisnitrobenzoic acid (DTNB) were purchased from the British Drug House (BDH, Dorset, UK). Hydroxylamine hydrochloride (H3NO·HCl), acetylthiocholine (ATC), and all other analytical grade reagents were
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purchased from Sigma Chemical Co. (St. Louis, USA). 2.1. EXPERIMENTAL PROTOCOL: Animals (n=12) (weight, 150-200 g) were randomly divided into two experimental groups (n=6); Control and Choline. Control rats received tap water daily via the oral route in a volume of 0.2 ml/150 g body weight. Test rats received an aqueous solution of choline bitartrate tablet powder via the oral route, at a dose of 52 mg/kg/day body weight daily in a volume of 0.2 ml/150 g body weight, for the duration of five weeks. The dose selection is equivalent to the recommended dose for humans (500 mg/day/60 Kg body weight) mentioned by the manufacturer, and previous reports (Zeisel and da Costa, 2009), and from a pilot study conducted in our laboratory (Tabassum and Haider, 2016). At the end of four weeks’ treatment schedule, behavior studies were performed as outlined in figure
ACCEPTED MANUSCRIPT 1. Behavioral tests included the Open Field test (OFT), the Inverted Screen test (KIST), the Beam Walking test (BWT) to assess locomotor activity, muscular strength and motor coordination, respectively, the Novel Object Recognition test (NORT), Morris Water Maze test (MWM) and the Passive Avoidance test (PAT) to determine recognition ability, spatial and associative learning and memory performance. After monitoring behavioral activities, rats were decapitated, and their brains were removed within 30 s and dissected to collect hippocampal tissue, as
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described previously (Haider et al. 2016).
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Fig. 1: Schematic representation of treatment schedule and Experimental design. 2.2. BEHAVIORAL PROTOCOLS:
Food Intake and Body Weight:
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2.2.1.
Food intake was monitored daily during the five weeks of treatment by giving rats a weighed amount of food
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and weighing the remaining food at next feeding. Body weights of the rats were also monitored daily during the five weeks of the treatment. Animals were weighed at the beginning of the experiment, and were followed up with daily until the end of this study. 2.2.2.
Assessment of Locomotor Activity: Following chronic choline supplementation, OFT, KIST, and BWT were performed to assess locomotor
activity, muscular strength and motor coordination, respectively.
Kondziela's Inverted Screen Test: Kondziela’s inverted screen test has been used previously for measure of muscular strength using all four limbs (Kondziela, 1964). Nearly all healthy animals easily score maximum on this task. The inverted screen is a 43cm square of wire mesh consisting of 12 mm squares of 1 mm diameter wire. It is bordered by a 4 cm deep wooden beading (which prevents animals which attempt to from climbing on
ACCEPTED MANUSCRIPT to the other side). The test was done by placing the rat in the center of wire mesh screen and the screen was rotated to an inverted position over 120 seconds with the rat’s head declining first. The time when the rat falls off from the screen was noted. Animals were scored inverted screen: for 2 min: Falling between 1-10 sec = 1, Falling between 11-25 sec = 2, Falling between 26-60 sec = 3, Falling between 61-90 sec = 4, Falling after 90sec = 5.
Beam Walking Test: Beam walking is a test of motor coordination (Goldstein and Davis, 1990). The rats have to cross a beam which is suspended between a start platform and their home cage at a height of 50 cm and is
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supported by two pillars. A cushion was placed under the beam to protect the animals from the bang into the
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floor. The difficulty of this task can be assorted by using beams with different shapes and widths (Jover et al. 2006). Motor coordination and balance was assessed by the ability of a rat to crossways a graded series of
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beams. Three circular beams of different diameter were used in this study such as 3 cm, 2 cm, 1 cm and length of 100 cm. In the training phase animals were trained to traverse the beam (from widest to narrowest) directly
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into the animal's home cage. This helps to make certain that the behavior during testing is more stable and more precisely reflects motor coordination as opposed to the rodent’s natural aversion to crossing over unprotected spaces. After training session testing phase was done, the time taken to cross the beam and number of foot slips
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off the beam was determined. The foot slips off the beam made by rat was scored on a scale of 0-6 as follows: 0 points, the rat was not able to stay on the beam, 1 point; the rat did not move but able to stay on beam, 2 points;
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the rat tried to traverse beam but fell, 3points; the rat traversed the beam with multiple slips (4-6), 4 points; the rat traversed the beam with few foot slips (2 or 3), 5 points; the rat traversed the beam with only one slip of the
2.2.3.
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hind limb, 6 points; the rat traversed the beam without any slip of hind limb (Puurunen, 2001). Assessment of Memory Performance:
The memory performance of rats following choline supplementation was determined across three domains i.e.,
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recognition, spatial and associative memory function by using NORT, MWM and PA tests, respectively. The details of apparatus used and procedures followed was essentially the same as described by Haider et al. (2016).
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2.3. OXIDATIVE AND NEUROCHEMICAL ANALYSIS: Oxidative status was analyzed according to Haider et al. (2015; 2016) to estimate lipid peroxidation (LPO),
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catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities, and GSH and protein content. ACh content and AChE activity was estimated by the same method as described by Batool et al. (2016) and Haider et al. (2015), respectively. Analysis of monoamines and their major metabolites [norepinephrine (NA), dihydroxyphenyl acetic acid (DOPAC), dopamine (DA), 5-hydroxyindoleacetic acid (5-HIAA), and 5 hydroxytryptamine (5-HT)] was performed in rest of the brain and the hippocampus using high-performance liquid chromatography with electrochemical detection (HPLC-EC), in the same manner as described by Haider et al. (2016). 2.4. STATISTICAL ANALYSIS:
ACCEPTED MANUSCRIPT All data are presented as the mean ± S.D. Statistical analyses were performed using SPSS software version 20.0 for windows. All the parameters were tested with the Shapiro-Wilks test and found to be normally distributed (p<0.05); therefore, in instances of multiple mean comparisons, the results of the behavioral and neurochemical assessment were analyzed by Student’s t-test for the two experimental groups for each parameter. Escape latencies during acquisition trials in MWM were analyzed by two-way ANOVA (repeated measures) followed by multiple comparisons by Bonferroni’s test. The level of significance for all comparisons was set at p≤0.05. 3.
RESULTS:
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The objective of this study is to investigate the effects of chronic choline supplementation in healthy adult
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rats on locomotor performance and learning and memory functions, along with determining changes in associated oxidative and neurochemical profiles in brain and hippocampus. These data may support future use of choline as a
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dietary supplement for improving locomotion and cognitive function. Food intake and body weight were not affected following all treatments as shown in table 1.
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TABLE 1: The effects of chronic choline intake on daily food intake and the body weight of animals before and after treatment. Data are presented as the mean ± SD (n = 6). A non-significant effect was obtained by the
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independent sample t-test when compared with control rats as well as with pretreatment values. FOOD INTAKE (g)
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CHOLINE
10.92±0.54
162.17±1.71
158.24±1.45
10.96±0.44
165.55±1.86
161.25±1.29
CHOLINE
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SALINE
BODY WEIGHT (g)
10.97±0.35
WEEK 2
10.97±0.41
WEEK 3
10.26±1.08
9.89±0.19
166.23±1.34
162.12±1.38
WEEK 4
10.33±0.34
10.21±1.72
165.63±0.92
160.14±0.97
WEEK 5
10.11±0.64
10.01±0.33
165.55±0.75
160.06±0.86
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WEEK 1
Effect of choline on the locomotor function of rats was evaluated via assessing locomotor activity,
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muscular strength and motor coordination by using OFT, KIST and BWT. The results showed that chronic choline supplementation improved the locomotor performance of adult rats. The results of OFT revealed significant
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improvement in locomotor activity evident by a significant reduction in movement latency (t = 4.092, p<0.01) from central area (fig. 2a), and a significant increase in square crossings in choline supplemented adult rats during five min time span, compared to control rats (t = -3.393, p<0.01; fig. 2b).
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Fig. 2: The effect of chronic choline administration on locomotor activity was evaluated by the open field test in terms of movement latency from the central square (A), and the number of squares crossed (B). For each group
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n=6; values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are
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expressed as *=p<0.05, **=p<0.01, compared to control. An analysis of the data obtained during KIST showed that following chronic choline supplementation,
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muscular strength of adult rats was improved, which was evident by a significant increase in fall latencies from
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the inverted screen in choline treated group compared to control group (t = -9.899, p<0.01; fig. 3).
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Fig. 3: The effect of chronic choline administration on muscular strength was evaluated by Kondzeila’s inverted screen test in terms of latency to fall from screen, which were then scored as reported in materials and methods. For each group, n=6; values are presented as the mean ± S.D. Significant differences were obtained
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by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control. Motor coordination assessed by BWT was also improved following chronic choline supplementation indicated by a significant reduction in latency to cross the beam (fig. 4) across three different diameters include 3 cm beam (t = 8.838, p<0.01), 2 cm beam (t = 8.598, p<0.01) and 1 cm beam (t = 7.727, p<0.01); no significant change was observed in scores for foot slips made during beam crossings.
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Fig. 4: The effect of chronic choline administration on motor coordination was evaluated by Beam walking
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test in terms of latency (sec) to cross the beam (A) of 3 different diameters (3 cm, 2 cm and 1 cm) without foot slips (B) while crossing the beam, which were then scored as reported in materials and methods. For each
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group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-
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test and are expressed as *=p<0.05, **=p<0.01, compared to control. The effects of chronic choline supplementation on learning and memory performance and cognitive ability was evaluated by NORT, MWM and PAT that are used to assess recognition, spatial reference and
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associative learning and memory performance, respectively. Recognition memory was assessed by ORT in terms of % exploration and discrimination index presented in fig. 5 (a and b). It was observed that recognition memory was improved in the choline treated group, evidenced by a significant decline in % exploration (fig. 5a) for familiar objects (t = 35.11, p<0.01) and a significant increase in % exploration for novel objects (t = 35.11,
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p<0.01), along with a significant increase in discrimination index (fig. 5b) (t = 39.338, p<0.01) in the choline
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treated group compared to the control group.
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Fig. 5: The effect of chronic choline administration on recognition memory was evaluated by the Novel object recognition task in terms of % exploration (A), and discrimination index (B). For each group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control.
ACCEPTED MANUSCRIPT The effects on spatial cognitive abilities of animals were determined using the MWM task. To find the difference in escape latencies of animals during four acquisition trials, two-way ANOVA (repeated measures) was performed. Our results indicated significant effect of trials (F(4,140)=847.142, p<0.01), groups (F(6,35)=50.465, p<0.01) and interaction between trials x groups (F(24,140)=16.787, p<0.01). Pair-wise comparisons by the Bonferroni test revealed that escape latencies of animals decreased significantly (p<0.01) over trials (shown in figure 6a), which may be due to familiarization to the environment and due to adaptation to the repeated training trials. Analysis of cumulative escape latencies of all acquisition trials revealed a significant decline (t = 6.676, p<0.01) in choline treated rats compared to control rats (figure 6b). Reference memory was
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examined by performing probe trials (free swimming without platform) in MWM tank after 1 hr. and 24 hr. of the last training trial, via monitoring the time spent in the target quadrant (NW) and the number of entries over
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the target quadrant, time taken to reach the target quadrant (quadrant latency), and time taken to reach the platform location (platform latency). Data analysis by Student’s t-test revealed significant increases in time
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spent in the target quadrant (t = 4.681, p<0.01), the number of entries over the target quadrant (t = 3.862, p<0.01), quadrant latency (t = 8.981, p<0.01) and platform latency (t = 8.019, p<0.01) during the 1 hr. probe trial (figure 6c) in the choline supplemented group compared to control group. Significant increases in time
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spent in the target quadrant (t = 5.314, p<0.01), number of entries over target quadrant (t = 6.01, p<0.01), quadrant latency (t = 7.274, p<0.01) and platform latency (t = 9.509, p<0.01) during 24 hr. probe trial (figure 6d) were observed in the choline supplemented group, compared to the control group. These results suggest that
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spatial reference memory acquisition and retention improved following chronic choline supplementation. b)
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Fig. 6: The effect of chronic choline administration on spatial memory performance of rats was determined in
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a spatial memory task [Morris Water Maze (MWM)]. (A) Escape latencies (sec) of rats in a spatial memory task during exposure to acquisition training trials, which were measured for 120 seconds for each trial. For each group n=6; values are presented as the mean ± S.D. (B) Averaged escape latencies (sec) for each animal in
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both groups are analyzed by Student’s t-test, and significant differences are presented as *=p<0.05, **=p<0.01, compared to control. During the probe trials performed after 1 hr. (C) and 24 hr. (D) after last acquisition trial, spatial memory performance was evaluated by monitoring duration of time spent (sec) in the
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target quadrant (NW) in absence of the platform, the number of entries made by each rat over the target quadrant (NW), time (sec) to enter into the target quadrant (Quadrant latency) and time (sec) to reach the platform location (platform latency) during duration of 120 seconds when the platform was removed. For each
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group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Students’ t-
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test and are expressed as *=p<0.05, **=p<0.01, compared to control. Associative memory was assessed by PAT in terms of the difference between pre-training and posttraining step-through latencies to enter the dark compartment in both acquisition and retention phases (fig. 7).
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Associative memory improved following treatment with choline, as evidenced by significant increases in the difference in step-through latencies during acquisition (t = 11.475, p<0.01) and retention phases (t = 40.277, p<0.01). Data analysis of step-through latencies evaluated during the acquisition phase by Student’s t-test
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revealed significant increases (t = 12.33, p<0.01) in step-through latencies of choline supplemented rats compared to control group as shown in figure 7. Similarly, Student’s t-test analysis for step-through latency evaluated during retention also showed significant increases (t = 26.19, p<0.01) in step-through latencies of choline supplemented rats compared to control group, as shown in figure 7.
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Fig. 7: The effect of chronic choline administration on avoidance memory performance of rats was assessed in
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the Passive avoidance test (PA) for 5 min by recording the step-through latencies (sec) during the initial training trial, during the acquisition phase 60 minutes after the initial training trial, and during the retention
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phase, 24 hr. after the initial training trial. The difference of step-trough latencies of the test trial (the time taken by rats to enter the aversive stimulus-associated dark compartment) to step-trough latency of initial training trial (the time required for rats to enter into the dark compartment) was computed for each animal as
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an index of memory deficit. For each group n=6; values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control.
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The effects of chronic choline supplementation on the brain oxidative profile were determined in terms of lipid peroxidation levels (MDA), antioxidant enzyme activities (CAT, GPX and SOD) and levels of antioxidant compounds (GSH and protein) in the brain, are shown in figure 8. The results show that following
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chronic choline supplementation, there was a significant decline in lipid peroxidation levels (MDA) (t = 6.348, p<0.01; fig. 8a), a marked increase in antioxidant enzymes (fig. 8b); GPx (t = 11.422, p<0.01), CAT (t = 12.248,
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p<0.01) and SOD (t = 5.477, p<0.01). There was also a significant increase in levels of antioxidant compounds (fig. 8c); GSH (t = 6.612, p<0.01) and protein content (t = 6.414, p<0.01) in brains of choline treated rats
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compared to controls.
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Fig. 8: The effect of chronic choline administration on the oxidative profile of rats was assessed via
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determination of the levels of lipid peroxidation (A), levels of antioxidant [GSH(nmol/g) and Protein(g/g)] (B) and levels of antioxidant enzymes [CAT (µmol/min/g), GPX (µmol/min/g), SOD (U/g)] (C). For each group,
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n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control.
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Levels of Acetylcholine (ACh) and its metabolizing enzyme acetylcholine esterase (AChE) in the brain were determined by spectrophotometric analysis. ACh levels were significantly increased (t = 5.77, p<0.01) in choline treated rats compared to the control group, as shown in figure 9a. AChE activity significantly declined (t
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= 7.574, p<0.01) in choline treated rats compared to the control group, as shown in figure 9b. These results suggest that brain ACh content was increased following intake of choline via reduced AChE activity. b)
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Fig. 9: The effect of chronic choline administration on the cholinergic profile of rats was assessed via determination of acetylcholine content (A) and ACHE activity (B). For each group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01, compared to control. The levels of monoamines (NA, DA and 5-HT) and their metabolites (DOPAC, HVA and 5-HIAA) were estimated using HPLC-EC in rest of the brain and hippocampal samples of both groups. Monoamine levels increased following choline treatment. An analysis of the NA levels showed a marked increase in rest of brain (t = 3.48, p<0.01) and hippocampus (t = 5.148, p<0.01) of choline-treated rats compared to control rats (figure 10a). Data analysis on dopamine and its metabolites; DOPAC and HVA by Student’s t-test revealed significant increase in DA levels in both in rest of brain (t = 2.23, p<0.05) and hippocampus (t = 6.957, p<0.01),
ACCEPTED MANUSCRIPT while its metabolite DOPAC was significantly increased in rest of brain (t = 4.33, p<0.01) and decreased in hippocampus (t = 4.696, p<0.01) while no significant change was observed in HVA levels in both rest of brain and hippocampus (figure 10b). Analysis of levels of 5-HT and its metabolite 5-HIAA showed that choline supplementation altered serotonin metabolism as there was a significant increase in 5-HT levels in both rest of brain (t = 10.081, p<0.01) and hippocampus (t = 8.249, p<0.01) and 5-HIAA levels were increased in rest of brain (t = 5.678, p<0.01) while decreased in hippocampus (t = 5.737, p<0.01) (figure 10c). These results suggest that following chronic choline supplementation neurochemical profile was improved as there was a marked increase in monoamine metabolism.
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Fig. 10: The effects of chronic choline administration on the monoamine profile of rats in rest of brain and hippocampus samples were assessed via determining the levels of NA (A), DA and its metabolites (DOPAC & HVA)
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(B), levels of 5-HT and its metabolite 5-HIAA (C). For each group, n=6, and values are presented as the mean ± S.D. Significant differences were obtained by Student’s t-test and are expressed as *=p<0.05, **=p<0.01,
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compared to control. DISCUSSION:
The aim of current study is to reveal that whether chronic choline supplementation could exert a beneficial
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effect on cognitive function and locomotor performance of healthy adult rats receiving choline at a dose equivalent to AI for the period of five weeks. Our findings show that chronic choline intake is beneficial in improving memory
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function and locomotor performance in healthy adult rats at an appropriate dose, in contrast to previous reports that observed no benefit of choline supplementation on memory function in healthy individuals (Lippelt et al. 2016) and
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in rats, but was only effective when administered in combination with caffeine (Nagrecha et al. 2013). However, our findings are consistent with an earlier study that reported beneficial effects of acute choline supplementation on
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motor performance (Naber et al. 2015). However, the effects on memory function in healthy adult rats and associated oxidative and neurochemical alterations were not addressed. Thus, our study may be the first to assess the beneficial effects of chronic choline supplementation on both memory function and locomotor performance in
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healthy young rats at a dose equivalent to AI. Studies show that dietary choline supplementation during development produces life-long modifications in the brain and subsequent improved behavioral performance, including structural, neurochemical, and electrophysiological alterations responsible for enhancement in cognitive functioning (Glenn et al. 2012; Meck and Williams, 2003; Schneider and Thomas, 2016). Moreover, as the precursor of acetylcholine, choline is also reported to improve cognitive performance in elderly patients (Leermakers et al. 2015). However, in healthy populations, the favorable effects of choline intake have not been observed. Differences in dosage and type of choline used may account for this (Leermakers et al. 2015). Hence, the question may be raised as to whether the effect of choline supplementation is dependent on age and health status. Thus, our study monitored the effects of choline intake in healthy adult rats rather than in diseased or aged rats. Previously we reported that memory function was improved
ACCEPTED MANUSCRIPT following chronic choline intake, via modulation of acetylcholine levels in the brain (Tabassum and Haider, 2016). The present study was designed to investigate the effectiveness of chronic choline supplementation in improving memory and locomotor performance, and in the modulation of oxidative and neurochemical status of a healthy brain. Consistent with our previous study, present findings show that along with inducing changes in behavioral performance, chronic choline intake at AI also has a positive effect on the oxidative and neurochemical status of the brain, indicating that dietary choline administration is beneficial for improving the physical and mental health of adult rats.
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In previous reports, choline is typically administered in diet or water (Borges et al. 2015; Guseva, 2008),
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but few studies administered choline orally (Borges et al. 2015; Wallace et al. 2012). Furthermore, very few studies used a choline dose that was equivalent to AI (Wallace et al. 2012). However, it is evident that the effects of choline
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are dependent upon its dosage (Borges et al. 2015), as smaller doses are not neuroprotective and high doses produce toxic effects such as hypo-activity, hypothermia, hypotension, cyanosis, and high mortality, while moderate doses
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are beneficial (Borges et al. 2015). Commercially available choline tablets (recommended for enhanced absorption and for treatment of liver disease) were used at a dose equivalent to AI (Wallace et al. 2012; National Academies Press, 1998) in our study. Previously, studies of oral choline administration in adults have administered either a
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single dose (Naber et al. 2015) or doses over seven days (Borges et al. 2015), while we orally administered choline daily for five weeks. Food intake and body weight of rats were monitored throughout the experiment. Changes in
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food intake and body weight of rats following choline supplementation were not significantly different than figures reported in previous studies (Leermakers et al. 2015; Killgore, 2010). Previously, some researchers have observed
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adverse effects following choline administration, so we monitored animals throughout the experiment for such effects as mortality and hypo-activity, but no adverse effects were noted in our study (Borges et al. 2015; Unal et al. 1998). Thus, these data suggest that choline administration at doses equivalent to AI is probably safe.
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Because choline is a component of both cell membranes and neurotransmitters involved in nerve signaling, it has a unique role in memory preservation and prevention of cognitive decline (Wood and Allison, 1982).
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Numerous studies report enhancement in rodents’ memory performance following choline intake (Naber et al. 2015; Leermakers et al. 2015). However, studies show positive effects on cognitive status with choline supplementation during pregnancy (Glenn et al. 2012; Borges et al. 2015; Meck and Williams, 2003) in elderly patients (Leermakers
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et al. 2015), or when given in combination with other compounds such as caffeine (Nagrecha et al. 2013), or when given during/after pathological conditions such as dementia (Naber et al. 2015; Blake et al. 2012), ischemia (Borges et al. 2015), traumatic brain injury (Borges et al. 2015; Guseva et al. 2008) or alcohol exposure (Schneider and Thomas, 2016) in rodents or in postmenopausal women (Wallace et al. 2012) but very few studies assessed choline effectiveness when given alone to healthy adults. A recent review reported that choline was not significantly beneficial in improving cognition in healthy patients (Leermakers et al. 2015); while a similar study also reported that choline was not effective when given alone in healthy subjects (Lippelt et al. 2016; Nagrecha et al. 2013). In contrast, our findings suggest that chronic choline administration is effective in improving memory performance of healthy adult rats. Our results showed that choline administration not only improved spatial reference memory but also improved recognition and associative memory performance in rats. This improvement might be because choline
ACCEPTED MANUSCRIPT is the precursor of ACh (Leermakers et al. 2015), so choline administration increases ACh synthesis and release in cholinergic neurons (Sarter and Parikh, 2005), which may be beneficial for neurological health of adult rats, or this might be due to increased availability of phosphatidylcholine that positively affects the neuronal membranes and in turn improves cognitive function (Leermakers et al. 2015; Conant and Schauss, 2004). Along with assessing cognitive function, we also assessed the locomotor performance of rats after choline intake because studies have shown that, besides cognition, cholinergic brain networks are also involved in motor functions (Naber et al. 2015; Woolf, 1991). Our findings show that locomotor activity, muscular strength and motor
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coordination, assessed by OFT, KIST, and BWT, were significantly improved following chronic choline
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administration. This improvement in locomotor performance was consistent with previous studies, which suggest a crucial role of choline in motor functions (Naber et al. 2015). Earlier studies reported enhancement in locomotor
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activity following an increase in choline release in hippocampus and cortex (Toide, 1989). Impaired motor coordination and motor learning after dietary choline deprivation (Pacelli et al. 2010), and attenuation of motor
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deficits by postnatal choline supplementation have also been shown earlier (Arazi et al. 2014). The probable mechanism responsible for improved locomotor performance is increased ACh synthesis in the brain following choline administration. It is reported that in the brain, choline-to-acetylcholine synthesis has an important role
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(Naber et al. 2015) that may affect the cholinergic motor neurons via increasing calcium release from sarcoplasmic reticulum, leading to muscle contraction (Lowes et al. 2013), and may increase motor coordination and locomotor
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activity.
In addition to observing behavioral changes following choline administration, the present study has also
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determined the effect of chronic choline administration on alterations in brain redox status and neurochemical changes in brain and hippocampus. Choline is part of the mitochondrial membrane, the main source of intracellular reactive oxygen species (ROS) and is required for cellular signaling. However, ROS can also cause cell damage, so
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a firm control by endogenous antioxidant scavenging systems exists that limits this damage (Lowes et al. 2013). These are comprised of antioxidant enzymes, including SOD, which catalyzes the dismutation of superoxide to
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H2O2, and CAT or GPX, which then decomposes H 2O2 to water (Dumont et al. 2009), and non-enzymatic antioxidants, such as GSH and protective proteins (Halliwell, 2011). Previously, it has been determined that oxidative stress was enhanced following dietary choline deprivation. This effect is mediated by increased ROS
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production and lipid peroxidation (increased MDA levels), decrease content of GSH and proteins, and reduced activities of SOD and CAT (Santos et al. 2015). The findings in the present paper showed that chronic choline administration reduces the oxidative stress as it reduces the production of ROS via decreasing the levels of lipid peroxidation, stimulating the activities of antioxidant enzymes (CAT, GPX and SOD) and by increasing the antioxidant stores of GSH and proteins. These results are consistent with the studies that reported a reduction in oxidative stress following choline administration in various pathological conditions (Mehta et al. 2010) via decreasing lipid peroxidation. It is evident that administered choline is incorporated into ACh, so by relating choline administration with central cholinergic activity, we found that brain acetylcholine concentrations increased following choline administration, consistent with previous reports showing declines in ACh content following dietary choline
ACCEPTED MANUSCRIPT deficiency (Wecker, 1979); but contrast with studies reporting that choline supplementation modulates ACh synthesis only in certain neurological disorders or after drug-induced neuronal activity, but under normal conditions remains unaltered (Wecker, 1986). In the present study, neurochemical alterations following chronic choline administration were also investigated. We observed that choline administration positively affected the activity of catecholaminergic and serotonergic neurons both in the whole brain and in hippocampus, as there was a marked rise in levels of noradrenaline, modulation of dopamine and serotonin metabolism in brain and hippocampus via increasing their synthesis and release in respective neurons. These alterations might occur via affecting the
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catecholaminergic neurons and by increasing the activity of tyrosine hydroxylase, as cholinergic drugs are known to
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enhance both dopamine synthesis and turnover (Ulus and Wurtman, 1976). However, in contrast, some studies report that choline administration did not alter neurochemical profiles (Wecker, 1979). Thus, our findings suggest
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that as the precursor of ACh, choline not only affects cholinergic neurotransmission but also modulates catecholaminergic and serotonergic neurotransmission leading to improvement in motor performance and memory
5.
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function. CONCLUSION:
The findings of the present study, together with previous findings, suggest that choline administration is
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beneficial for neurological health of not only children or elderly patients but also healthy adults, in particular improving cognitive and locomotor performance. In addition, chronic choline intake was found to improve
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behavioral, oxidative and neurochemical outcomes in the normal population. Although choline supplements are commercially available, these are considered ineffective in improving brain function, particularly memory and
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motor performance. Thus, our current findings suggest that chronic choline administration at doses equivalent to AI might have a positive impact on brain function, particularly on cognitive and motor function in healthy adult rats. These findings suggest the use of available choline supplements for the neurological functioning in humans may be
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beneficial. Thus, it may be suggested that in the future choline may be used as a safe and effective supplement for improving the neurological health status of normal individuals and that it might also be beneficial in preventing
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cognitive and motor disorders later in life. ACKNOWLEDGEMENTS:
English. 7.
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Authors are grateful to Prof. Hafiz Syed Ali Athar for critically going through the manuscript for improving the
FUNDING AND DISCLOSURE: The funds for experimental material and animals for the present study were provided by Higher Education
Commission (HEC), Pakistan and University of Karachi, Karachi, Pakistan. Authors have no competing financial interests in relation to the work described. 8.
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Graphical abstract
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HIGHLIGHTS Study was aimed to investigate whether choline intake is beneficial in healthy adults.
Chronic choline intake at an adequate dose improved brain functioning in healthy adults
Chronic choline administration improved cognitive and locomotor function.
Choline reduced oxidative stress, enhanced cholinergic and monoaminergic transmission.
Hence, choline tablets may be suggested as a safer and effective supplement to improve neurological
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health.