Differential effects of early-life NMDA receptor antagonism on aspartame-impaired insulin tolerance and behavior

    Differential effects of early-life NMDA receptor antagonism on aspartameimpaired insulin tolerance and behavior Kate S. Collison, Angela Inglis, Sherin Shibin, Bernard Andres, Rosario Ubungen, Jennifer Thiam, Princess Mata, Futwan A. Al-Mohanna PII: DOI: Reference:

S0031-9384(16)30816-2 doi:10.1016/j.physbeh.2016.09.011 PHB 11491

To appear in:

Physiology & Behavior

Received date: Revised date: Accepted date:

25 April 2016 9 August 2016 13 September 2016

Please cite this article as: Collison Kate S., Inglis Angela, Shibin Sherin, Andres Bernard, Ubungen Rosario, Thiam Jennifer, Mata Princess, Al-Mohanna Futwan A., Differential effects of early-life NMDA receptor antagonism on aspartame-impaired insulin tolerance and behavior, Physiology & Behavior (2016), doi:10.1016/j.physbeh.2016.09.011

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Differential effects of early-life NMDA receptor antagonism on aspartame-impaired insulin tolerance and behavior

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Kate S. Collison, Angela Inglis, Sherin Shibin, Bernard Andres, Rosario Ubungen, Jennifer Thiam, Princess Mata, & Futwan A. Al-Mohanna.

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Department of Cell Biology, King Faisal Specialist Hospital & Research Centre, PO BOX 3354, Riyadh 11211, Saudi Arabia.

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Key words: aspartame, insulin tolerance, NMDA receptor antagonism, behavior, gut brain axis.

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Corresponding author and reprint requests: Kate S Collison PhD FRSC FRSB Department of Cell Biology King Faisal Specialist Hospital & Research Centre P. O. Box. 3354, Riyadh 11211 Saudi Arabia Tel. () 9661 442 7841 Fax. () 9661 442 7854 Email. [email protected]

Abbreviations ASP: aspartame; NNS: non-nutritive sweeteners; CGP: CGP 39551; CON: control; ITT: insulin tolerance test; AUC: area under the curve; NMDA: N-methyl-D-aspartate; NMDAR: N-methyl-D-aspartate receptor; CNS: central nervous system; NPY: neuropeptide Y; AgRP agouti-related peptide; CART: cocaine and amphetamine regulated transcript; POMC: proopiomelanocortin precursor; MCH: melanin concentrating hormone; CRR: Counter-regulatory response; STM: short term memory; LTM: long term memory; GSIS: glucose-stimulated insulin secretion. OF: Open Field; MWM: Morris Water Maze; PCA: principal component analysis;

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ACCEPTED MANUSCRIPT Abstract. We have previously showed that lifetime exposure to aspartame, commencing in utero via the mother’s diet, may impair insulin tolerance and cause behavioral deficits in adulthood via mechanisms which are

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incompletely understood. The role of the CNS in regulating glucose homeostasis has been highlighted by recent delineation of the gut-brain axis, in which N-methyl-D-aspartic acid receptors (NMDARs) are

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important in maintaining glucose homeostasis, in addition to regulating certain aspects of behavior.

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Since the gut-brain axis can be modulated by fetal programming, we hypothesized that early-life NMDAR antagonism may affect aspartame-induced glucose deregulation in adulthood, and may alter

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the aspartame behavioral phenotype. Accordingly, C57Bl/6J mice were chronically exposed to aspartame commencing in utero, in the presence and absence of maternal administration of the

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competitive NMDAR antagonist CGP 39551, from conception until weaning. Drug/diet interactions in adulthood glucocentric and behavioral parameters were assessed. Aspartame exposure elevated blood glucose and impaired insulin-induced glucose disposal during an insulin tolerance test, which could be

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normalized by NMDAR antagonism. The same effects were not observed in control diet mice, suggesting

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an early-life drug/diet interaction. Behavioral analysis of adult offspring indicated that NMDAR antagonism of control diet mice caused hyperlocomotion and impaired spatial navigation. Conversely

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hypolocomotion, reduced exploratory activity and increased anxiety-related behavior was apparent in aspartame diet mice with early-life NMDAR antagonism. Conclusion: significant drug/diet interactions in glucocentric and behavioral parameters were identified in aspartame-exposed mice with early-life NMDAR antagonism. This suggests a possible involvement of early NMDAR interactions in aspartameimpaired glucose homeostasis and behavioral deficits.

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ACCEPTED MANUSCRIPT 1. Introduction. Epidemiological studies have linked consumption of non-nutritive sweeteners (NNS) with a counterintuitive positive association with weight gain [1], the metabolic syndrome and type 2 diabetes [2-4].

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Consumption of NNS is increasing globally, and available evidence suggests that consumers of so-called ‘diet’ beverages are typically characterized as young to middle-aged, with a slight preponderance of

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females [5]. Within this demographic, little is known about the rate of consumption of NNS during

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pregnancy, or the long-term metabolic and behavioral outcomes for the offspring [6]. Aspartame (Laspartyl phenylalanine methyl ester) is one of the most widely available NNS, approved for use in more than 6000 dietary products consumed by the general public including pregnant and lactating women,

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children and adults [7,8]. However, a recent prospective study of > 3000 mothers and their children reported a doubling of the risk of being overweight amongst 1-year olds whose mothers consumed NNS-

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sweetened beverages daily during pregnancy [9]. Therefore there is mounting evidence that exposure to NNS either prenatally or at a later stage of life is associated with an increased risk of being overweight. In a study that was designed to mimic patterns of aspartame consumption in the human

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population, we have previously shown that chronic consumption of aspartame approximating the

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Acceptable Daily Intake limit [10] by C57Bl/6J mice commencing in utero via the mother’s diet, increased male adult offspring body weight and impaired insulin-stimulated glucose disposal during an insulin

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tolerance test [11]. We also showed that aspects of adulthood spatial cognition were impaired by the aspartame exposure. Additionally hyperglycemia together with alterations in feeding and increased anxiety-type behavior have recently been induced in adult rats exposed to aspartame prenatally via the mother’s diet [12]. Other rodent studies have demonstrated that exposure to aspartame in utero during pregnancy [13], and extending to include the lactation period and through to adulthood [14] results in a variety of metabolic, physiological and behavioral deficits in the mature adults. Recent studies have highlighted the importance of the central nervous system (CNS) in regulating glucose homeostasis via the bi-directional gut-brain axis [15]. Insulin-responsive neuroendocrine circuitry in the CNS limbic system, particularly in the hypothalamus, coordinates a multitude of physiological processes including the regulation of glucose homeostasis, insulin secretion and sensitivity, satiety and food intake [16]. Within the arcuate (ARC) nucleus of the hypothalamus, two main populations of neurons continuously monitor signals reflecting energy status, and coordinate the appropriate behavioral and metabolic responses. One neuronal population produces two appetitestimulating peptides: neuropeptide Y (NPY) and agouti-related peptide (AgRP); whilst the second 3

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express

several

appetite-suppressing

peptides:

α-MSH

derived

from

the

proopiomelanocortin precursor (POMC), and cocaine and amphetamine regulated transcript (CART) peptide [17]. Both hypothalamic neuronal populations also express functional insulin and leptin

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receptors which in turn regulate gluconeogenesis and glucose uptake by peripheral tissues via autonomic efferent neuroendocrine circuitry [18]. Importantly, there is increasing evidence that these

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systems controlling energy homeostasis in adulthood are vulnerable to metabolic imprinting in utero

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and in the case of rodents, during lactation and early postnatal life [19, 20].

In addition to the metabolic control exerted by peripheral hormones, hypothalamic neurons are also regulated by synaptic inputs; and glutamatergic signaling acts via multiple ionotropic receptors,

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including the N-methyl-D-aspartic acid (NMDA), D,L-α-amino-3-hydroxy-5-methyl-isoxazole propionic acid (AMPA), and kainic acid subtypes, in order to regulate energy homeostasis and aspects of behavior

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[21]. Hypothalamic AgRP and POMC neurons express ionotropic NMDA receptors (NMDARs); and research by Lam et al [22] has highlighted the importance of NMDARs in the forebrain-hindbrain

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neuronal circuitry regulating hepatic gluconeogenesis. Stimulation of hypothalamic paraventricular nucleus NMDA receptors has been shown to raise plasma glucose levels [23]; and intracereboventricular

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injections of NMDA can increase hepatic glucose production [24]. Functional NMDARs are also expressed on a wide spectrum of non-neuronal cells, where they are understood to have multiple roles

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in regulating insulin secretion and maintaining blood brain barrier integrity, to name but a few [25]. These observations demonstrate the critical role NMDARs play in modulating glucose homeostasis, which appears to be disrupted by aspartame exposure both in utero [11, 12] and during adulthood [26, 27].

Concomitant with their involvement in the neuroendocrine control of glucose and energy homeostasis, NMDARs are also centrally involved in learning & memory acquisition and modulation via the induction of long term potentiation [28]. The expression of NMDA receptor subunits is developmentally regulated, and changes in expression patterns have been shown to correlate with critical periods of CNS development [29], which is vulnerable to fetal programming. It is therefore apparent that NMDARs are critically involved in the regulation of both glucose and energy metabolism as well as many aspects of behavior, learning and memory, which can be modulated by aspartame exposure either during early life or in adulthood [11-13]. The hitherto unexplored possibility therefore exists that NMDARs may be involved in the effects of prenatal and postnatal exposure to aspartame. Studies designed to characterize the involvement of NMDARs during development and in the mature 4

ACCEPTED MANUSCRIPT CNS have involved the in vitro and in vivo use of NMDAR agonists, antagonists and receptor subunit gene knock-out models [30]. For example, the orally active competitive NMDAR antagonist CGP 39551 [31] has been utilized in studies investigating the effect of neonatal NMDAR blockade on adult

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locomotor activity and neurochemistry [32,33].

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Because metabolic and behavioral phenotypes are influenced by environmental variables during perinatal development, we hypothesized that NMDAR antagonism during early life might

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differentially modulate aspartame-induced glucose deregulation and the behavioral changes previously observed [11,12]. We elected to focus on the effect of early-life aspartame exposure on male offspring

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physiology since males appear to exhibited more adverse reactions than females in some [8,12] but not all [13,14] experimental paradigms, and because limiting our analysis to parameters in the male

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offspring may facilitate the elucidation of causal mechanisms. Accordingly, in order to gain insight into the potential involvement of NMDARs, male C57Bl/6J mice were chronically exposed to aspartame (46 mg/Kg/dy) commencing in utero via the mother’s diet, in the presence or absence of maternal

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administration of CGP 39551 (5mg/Kg/dy p.o.). Since several studies have demonstrated that prenatal or

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neonatal aspartame exposure causes deleterious effects in adulthood, intervention with the NMDAR antagonist was limited only to the prenatal and neonatal period, during which time modulation of

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NMDAR function is known to affect adulthood physiology and behavior, such as is the case with the hypothalamic model of obesity [34-36]. Drug/diet interactions in physiological parameters, behavior and spatial navigation variables in the adult male offspring were assessed.

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ACCEPTED MANUSCRIPT 2. Materials and Methods. 2.1. Animals and treatments.

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C57BL/6J mice of both sexes were obtained from the Jackson Laboratory (Maine, USA) and housed 3/cage in a controlled environment (pathogen-free conditions of 12 h light/dark cycle, 22 ± 2°C) with

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standard chow and water available ad libitum. The breeding and care of the animals were in accordance

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with the protocols approved by the Animal Care and Use Committee of the King Faisal Specialist Hospital & Research Centre. In order to obtain F1 offspring following prenatal and neonatal NMDAR antagonism,

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female breeders were randomly divided into four groups: Control diet (CON), Control diet + CGP 39551 (CON+CGP), aspartame diet (ASP), and aspartame diet + CGP 39551 (ASP + CGP). Aspartame (Sigma Aldrich, MO, USA) and the orally-active water-soluble drug CGP 39551 (Tocris Bioscience, MI, USA) were

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administered in the drinking water as the only source of water at 46mg/Kg bw and 5mg/Kg body weight respectively. The dose of CGP 39551 was selected on the basis of previous experiments demonstrating

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potent biological activity following oral administration [37]. Tolerability to the separate and combined interventions were previously ascertained in a pilot study which closely monitored the growth rate, food

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and fluid intake, and general well-being of the animals following exposure to the interventions for up to

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20 weeks of age (data not shown). After a 3-week period of acclimatization, female dams were placed with an experimentally-naïve male, in a ratio of 2 to 1, in order to mate. Animals were subsequently monitored and approximately 7 days prior to delivery, the dams were isolated and the day of parturition was defined as postnatal day 0 (P0). Dams continued to receive the interventions in the drinking water until weaning was completed at P28, whereupon male F1 offspring (no more than 3 animals per litter) in the CON and CON+CGP group were given ad lib plain drinking water and standard chow, and the offspring in the ASP and the ASP+CGP group were given ad lib drinking water containing 46 mg/Kg bw aspartame together with standard chow for the duration of the study. Animals were weighed at 7-week intervals. Food and fluid intake was measured at 7 weeks of age as previously described [11]. A schematic indicating an overview of the experimental design and interventions is presented in Figure 1. 2.2.

Behavioral testing.

Behavioral testing was performed between 14 and 16 weeks of age during the light period between 08:00 and 17:00h under dim light illumination. Mice were 14 weeks of age at the start of the behavioral

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ACCEPTED MANUSCRIPT experiments, and were subject to all tests performed in the order listed below. Mice were habituated in the testing room for 45 min prior to the commencement of each behavioral experiment.

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2.2.1. Open Field test. In order to measure the treatment effects on exploratory and locomotor parameters, mice were subject

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to the open field test at 14 weeks of age [38]. The apparatus consisted of a box (floor: 60cm x 60cm of white PVC; walls 30 cm high of clear Plexiglas). Mice were initially placed on one side of the arena, and

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the central zone was defined as the central 20cm x 20cm area. Locomotor and zone tracking data over a period of 5 minutes was collected using Noldus EthoVision XT 7.0 video-tracking system. Measured

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behavioral parameters were: maximum velocity (m/sec), mean velocity (m/sec), total mobility (sec), total distance (cm), duration of immobility (sec), duration of high mobility (sec), duration of contracted,

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normal and stretched posture (sec), rearing frequency, total vertical activity, total sectors crossed, duration of central and peripheral zone occupation (sec) and frequency of central and peripheral occupancy. The apparatus was cleaned between each mouse test session using 70% alcohol. Mice that

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remained immobile for 70% of the total test time were excluded from analysis. 2.2.2. Light / Dark Transition test.

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A light/dark transition test [39] was employed to further investigate the effects of drug/diet intervention on anxiety-related parameters. A 60 × 60 × 30 cm arena consisted of two compartments: a small chamber comprising one third of the area consisting of an inverted custom-made box painted black and termed the dark chamber. The remaining two-third of the area was brightly illuminated and termed the light chamber. (Illuminated side: 400 lux; dark side : 0.4 lux). Mice could cross freely from one compartment to the other via a small hole in the wall (5 cm height, 8cm width). Mice were placed in the light side facing the hole and observed using tracking software for 5 minutes during which the time spent in each of the two compartments was recorded. 2.2.3. Novel Object Recognition test. A novel-object recognition test was used to evaluate exploratory behavior and nonspatial working memory [40]. Mice which had previously been habituated in the OF test were allowed to explore two identical objects placed in the arena for 5 minutes (sample trial). Animals were returned to their cages during the inter-trial interval. The apparatus was cleaned between each mouse test session using 70% alcohol. The effects of drug/diet manipulation on short term memory (STM) was tested four hours later, 7

ACCEPTED MANUSCRIPT when one of the two familiar objects was replaced with a novel object and the mice were again allowed to explore for 5 minutes (STM test). All combinations and location of objects were used to prevent bias due to preference for a particular object or location. Exploration time was computed when the snout

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pointed to the object at a distance ≤ 4 cm. Mice were returned to their cages and 24 hours later, long term memory was tested in a paradigm where they were again presented with one familiar object from

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the STM test together with a 3rd novel object to explore in the apparatus (LTM test). Discrimination where, Tn = the amount

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index (DI) scores were calculated using the formula:

of time explored the novel object and Tf = the amount of time mice explored the familiar object. Mice

and analysis.

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2.2.4. Morris Water Maze (MWM) Testing.

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that did not explore any of the objects within the 5-min period were excluded from further experiments

Spatial navigation and place learning was assessed at 16 weeks of age in a MWM task, as previously

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described (11). Briefly, the apparatus consisted of a white circular pool of 150cm diameter and 60 cm

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height, filled with water made opaque by the addition of a small amount of non-toxic white paint (15 cm deep) and maintained at 24-26 oc. A circular escape platform (11 cm in diameter) was placed in a fixed

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South-West location hidden 0.5cm below the surface of the water, and 4 fixed-position geometric visual cues were kept in the room throughout the period of data acquisition. A digital camera was positioned above the center of the tank and linked to a tracking system (HVS Image Analysis VP-200, HVS Image, Hampton, UK) in order to record escape latencies, time spent swimming close to the location of the escape platform, swim speed and floating time (defined as percentage of time spent swimming with a speed of less than 0.06 m/sec), for each trial. The percentage of time spent in swimming parallel to the pool wall within a 16-cm distance from the wall (% thigmotaxis) was also recorded. Mice were given four consecutive days of acquisition training blocks that consisted of four trials per day with an inter-trial interval of 20 minutes. In order to investigate the effects of drug/diet interactions on allocentric spatial reference memory acquisition, the position of the hidden platform remained fixed and the entry point was pseudo-randomly selected from one of four compass locations each day, and the same sequence of starting points was used for all the mice tested. Mice were given 120 seconds to find the platform and if the mice failed to locate the platform within this period, the subject was gently guided onto the platform. All mice were allowed to rest on the platform for a 30 second interval after each trial. At the end of the training block, mice were placed in a pre-warmed drying cage prior to being returned to their 8

ACCEPTED MANUSCRIPT experimental cages. Experimental subjects that failed to show any significant improvement in escape latency during the 4 days of acquisition training in the MWM were excluded from the study. A probe test was run on day 5 after 4 days of acquisition. Before the probe test, the hidden platform was

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removed and the mouse was introduced from the quadrant opposite to the escape quadrant, where it

former location of the submerged platform were recorded. Random fed Insulin Tolerance test (ITT).

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2.3.

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was left to search for the platform for 60s. Time spent in the target quadrant and proximity to the

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Changes in the response to exogenous insulin challenge due to treatment effects were assessed by an ITT performed at 18 weeks of age. To avoid the confounding effects of exercise during behavioral testing on glucose homeostasis [41], plasma corticosterone [42] and NMDA receptor expression [43],

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treatment-identical exercise-naïve mice were used for the ITT and other related measurements of body characteristics. Prior to the commencement of the ITT all animals had full access to food and water. A

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tail vein baseline glucose reading was established before an intraperitoneal injection of insulin (Sigma,

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IL) at a dose of 0.75 U/kg body weight was administered, and whole blood glucose levels were measured at 15, 30, 45 and 60 minutes after injection as previously described (11). Assessment of insulin tolerance

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was made after calculating the Area Under the Curve for glucose (AUC

GLUCOSE),

the rate of glucose

utilization (KITT), and the half-life of glucose levels (T1/2). AUCs were calculated using the trapezoidal rule. KITT, defined as the percentage decline in glucose per minute, was calculated from the natural log (Ln) of glucose concentrations between time t1 and t2, formula

. The serum T1/2

defined as the time in minutes required for the glucose concentration to be halved, was calculated as: T1/2 =

[44].

At the conclusion of the study (21 weeks of age), overnight fasted animals were humanely euthanized with a mixture of xylazine and ketamine (10 mg/kg and 100 mg/kg respectively) and blood was collected from the inferior vena cava for further analysis. The white adipose tissue epididymal fat pads were carefully dissected out, rinsed twice in PBS buffer, blotted dry and weighed to the nearest 0.001 g. These tissues were snap-frozen for future studies. Concomitantly, the interscapular fat, and pancreas were similarly dissected, weighed and stored. ELISAs were used to detect changes in plasma insulin ( Crystal Chem Inc., IL; Cat# 90080) and serum corticosterone (Enzo Life Sciences cat# ADI-900-097).

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ACCEPTED MANUSCRIPT 2.4.

Statistical analysis.

Data was presented as means ± SEM for body characteristics, ITT and all the behavioral tests. A p value of ≤ 0.05 was considered to be statistically significant. Statistical analyses were performed using IBM

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SPSS statistics software version 20 (SPSS Inc., Chicago, IL). Body variables such as body weight, white adipose tissue, interscapular fat, and pancreas weights, glucose parameters, plasma Insulin and

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corticosterone were analyzed using two-way ANOVA for diet*drug effect, followed by a two-tailed

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Independent t-test to find significant effects between drug/diet groups. For the open field and object recognition test analyses, all the variables were computed by the EthoVision software using a custom tracking algorithm. Data collected were analyzed for significant drug/diet effects using independent t-

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test. Univariate ANOVA was used to interpret significant effect between drug and diet as factors. For the MWM test analysis, the following variables were identified: latency to reach platform (s), time spent in

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platform location (s), Thigmotaxis (%time) and floating time (%time). Data was calculated by the software by setting the threshold for float at speed <0.025m/s and thigmotaxis at 0.8%. The drug/diet

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and combined effects were examined using independent t-tests and two-way ANOVA, respectively. When, necessary, significant differences between factors were confirmed post-hoc with a Tukey’s test.

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To further understand commonalities in behavioral strategies which the mice used in the different behavioral tests, principal component analysis (PCA) was performed. Based on Pearson correlation

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coefficients (r) that were calculated, strongly related variables were removed from the analysis to avoid multicollinearity. Variables that correlated less than 0.70 were selected for PCA. Following that, variables that loaded with less than 0.400 communalities were excluded further from analysis.

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ACCEPTED MANUSCRIPT 3.

Results.

3.1. Effect of diet and early-life NMDAR antagonism on animal characteristics. Table 1 shows the effect of diet and early-life intervention with the CGP 39551 drug on food and fluid

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intake together with body characteristics. Mean aspartame intake was 45.44 ± 1.16 mg/Kg bw and 46.59 ± 0.68 mg/Kg bw in the aspartame diet mice (ASP) and the ASP + CGP group respectively. Food and fluid

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intake was unaffected by either diet or CGP 39551. ASP diet mice had elevated body weight [t(34)=-

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4.359; p=0.000], adipose tissue [t(34)=-3.082; p=0.004] and pancreas weight [t(34)=-2.583; p=0.014]; together with higher random-fed glucose levels [t(34)=-3.030; p=0.005] compared to CON diet mice.

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Whereas NMDAR antagonism in control-fed mice did not significantly affect body or adipose tissue mass, mice in the ASP+CGP group had significantly lower body [t(34)=2.739; p=0.010] and adipose tissue weight [t(34)=3.605; p=0.001] together with lower random-fed glucose levels [t(34)=2.162;

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p=0.038] and higher serum corticosterone levels [t(28)= -2.128; p=0.042] compared to ASP fed mice without NMDAR antagonism. A 2-way ANOVA for drug/diet interactions found significance for body

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weight [F(3,68)=7.834; p=0.007], adipose tissue weight [F(3,68)=8.308; p=0.005], and random-fed

3.2.

Insulin tolerance test.

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glucose levels [F(3,66)=7.011; p=0.010].

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Results of an ITT administered at 18 weeks of age are shown in Figure 2. As reported previously (8), ASP diet mice had an elevated glucose response to insulin challenge which persisted for the duration of the test [ T0: t(34)=-3.030, p=0.005; T15: t(34)=-3.416, p=0.002; T30: t(34)=-2.683, p=0.011; T45: t(34)=-3.390, p=0.002; T60: t(34)=-4.028, p=0.000)] (Figure 2A). In the absence of aspartame exposure, maternal CGP 39551 administration significantly raised insulin-stimulated glucose levels only towards the latter half of the test period [T0: t(34)=-1.570, p=0.126; T15: t(34)=-1.389, p=.174; T30: t(34)=-0.178, p=0.860; T45: t(33)=-2.136, p=0.040; T60: t(34)=-2.612, p=0.013] (Figure 2B); whereas ASP+CGP group mice exhibited a significantly enhanced clearance of glucose from the bloodstream in response to exogenous insulin [T0: t(34)=2.162, p=0.038; T15: t(34)=3.069, p=0.004; T30: t(34)=2.511, p=0.017, T45: t (31) =2.875, p=0.007; T60: t (34) 2.774, p=0.009] (Figure 2C). Area Under the Curve (AUC) analysis confirmed that NMDAR antagonism of ASP diet mice resulted in a more rapid hypoglycemic effect of insulin [F(3,68)=12.401, p=0.001] (Figure 2D). Further analysis showed that ASP diet mice had a lower first-order glucose rate constant (K ITT) and a longer glucose half-life following the insulin injection (Table 2, P≤0.05), indicating a greater degree of insulin resistance in ASP diet mice.

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ACCEPTED MANUSCRIPT 3.3.

Effect of diet and early-life NMDAR antagonism with CGP 39551 on behavior.

3.3.1. Open Field test analysis. Significant differences were identified in 14 out of 18 Open Field variables in ASP fed mice with early-life

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exposure to CGP 39551 (Table 3). Mice in the ASP+CGP group were more immobile than either ASP diet [t(34)=-2.471;p=0.019] or control diet mice in the absence of NMDAR antagonism [t(34)=-2.990;

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p=0.005]. Mean velocity, duration of movement, duration of highly mobile behavior and total distance

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covered were all lowest in the ASP+CGP diet group (P≤0.05). ASP diet mice with NMDAR antagonism also spent significantly more time assuming a contracted posture; and vertical activity, indicative of exploratory behavior and risk assessment was reduced in these mice. A reduction in time spent in the

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centre of the field, together with an increase in duration spent in the periphery indicated an anxiogenic trait in ASP+CGP mice who also entered the central zone significantly less frequently than ASP

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[t(34)=2.747; p=0.010] or CON mice [t(34)=3.985; p=0.000]. Self-grooming frequency was elevated only in the aspartame diet mice. Conversely, mice in the CON+CGP group had significantly higher maximum

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velocities [t(33)=-2.047; p=0.049], greater duration of hypermobility [t(32)=-2.240; p=0.032] and spent more time adopting a stretched posture [t(32)=-2.108; p=0.043]. Exploratory behavior (frequency and

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duration of central zone occupation) and markers of anxiety were unaffected by CGP 39551 in control mice. 2-way ANOVA revealed significant drug/diet interactions for maximum [F(3,67)=7.373; p=0.008]

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and mean velocity [F(3,68)=6.550; p=0.013]; duration of immobility [F(3,68)=6.558, p=0.013], high mobility [F(3,66)=10.809, p=0.002] and total mobility [F(3,68)=6.558; p=0.013]; duration of stretched [F(3,64)=9.456; p=0.003] and vertical postures [F(3,68)=5.628; p=0.021]. In summary, in the absence of aspartame exposure, NMDAR antagonism of control diet mice resulted in hyperlocomotion and no effects on exploratory / anxiety-related behavior. Conversely ASP diet mice treated with CGP 39551 exhibited an anxiogenic, hypolocomotive phenotype with reduced exploratory behavior. There was considerable evidence of drug/diet interactions in behavioral variables between the ASP and the ASP+CGP group mice. 3.3.2.

Light/Dark transition test.

The effect of drug/diet intervention on anxiogenic-type behavior was further assessed in the light / dark transition test. Mice in the ASP diet group spent more time in the dark compartment compared to controls, suggesting increased anxiety although this observation fell just short of significance (Figure 3A, t(28)=-1.999; p=0.055). There was no effect of drug on dark compartment occupancy.

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ACCEPTED MANUSCRIPT 3.3.3. Object recognition test. The effect of drug/diet intervention on exploratory behavior and object recognition is shown in Figure 3. Animals were given a sampling session with two identical objects, followed by one short term memory

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test 4 hours later (STM test) and a long term memory test 24 hours later (LTM test). Total exploratory time in the training session was significantly shorter in the ASP diet mice compared to controls (Figure

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3B, t(32)=2.923; p=0.006). Four hours later, the STM test indicated that the lower object exploratory

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time in ASP diet mice was statistically non-significant [t(29)=1.299; p=0.204], whereas mice in the ASP+CGP group spent significantly less time exploring the objects [t(32)=3.055;p=0.005]. The effect of diet/drug intervention on object recognition memory as assessed by the discrimination index (DI) is

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presented in Figure 3C. During the short term memory (STM) test, mice in the ASP+CGP group showed significantly greater interest in the novel object despite spending less total time exploring the objects

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[t(25)=-3.019; p=0.006]; whereas mice in the ASP diet group seemed to show significantly less interest in the novel object during the LTM test (P≤0.05). Taken together these results indicate a reduction in

Open Field test.

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3.3.4. Morris Water Maze test.

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exploratory behavior in the ASP+CGP, consistent with the reduced exploratory behavior observed in the

We have previously shown that aspartame exposure can affect spatial navigation and learning during

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the Morris Water Maze (MWM) test [11]. In the present study, Figure 4A indicates the effect of drug/diet interventions on cued escape latencies over 4 days of acquisition training blocks, consisting of 4 trials per block. All mice improved their performance over the 4 days of acquisition training, however by the 3rd day all 3 drug/diet groups had significantly higher escape latencies, indicative of a poorer learning performance in the CON+CGP [t(34)=-3.404; p=0.002], ASP [t(34)=-2.161; p=0.038] and ASP+CGP [t(34)=-4.362; p=0.000] groups compared to controls (P≤0.05). Area under the curve (AUC) analysis of escape latencies across all blocks are shown in Figure 4B, which indicated a significant impairment in MWM spatial navigation in the CON+CGP [t(34)=-3.149; p=0.003], ASP [t(34)=-3.292; p=0.002] and ASP+CGP [t(34)=-3.460; p=0.001] groups compared to controls (CON). Swim speed was unaffected by either diet or drug treatment (data not shown). As testing progressed, mice learned to spend more time searching closer to the platform. Differences in mean proximity to the center of the escape platform between the 4 drug/diet groups is shown in Figure 4C. Mice in the CON group spent significantly more time swimming closer to the center of the platform on Day 2 [t(34)=2.392; p=0.022] and Day 3 [t(34)=3.598; p=0.001] of the acquisition 13

ACCEPTED MANUSCRIPT training. Thigmotactic behavior, as defined by the mean percentage of time the mice spent swimming close to the walls of the tank, is shown in Figure 5. Although mice initially swim close to the walls of the tank in order to escape the water, this behavior generally resolved into a search for the escape platform

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over repeated trials (Figure 5A). AUC analysis of % thigmotaxis indicated that mice consuming the aspartame diet, either in the presence or absence of maternal administration of CGP 39551, retained a

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higher level of thigmotactic behavior which could be indicative of anxiety-type behavior (Figure 5B).

acquisition training (Figure 5C).

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Non-directional floating behavior in control mice accounted for 12% of the time during the 4 days of Floating behavior in the ASP and ASP+CGP group mice was

approximately twice that of controls on Day 2-4 [F(3,68)=3.820, p=0.014; F(3,68)=3.022, p=0.036;

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F(3,66)=2.967, p=0.038]; and AUC analysis revealed significantly greater overall floating behavior in these groups [F(3,68)=2.844; p=0.044] (Figure 5D) which, together with the increased thigmotactic

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behavior, could account for the higher escape latencies seen in the ASP and ASP+CGP group mice. During the probe test administered on Day 5, we found no significant differences in mouse target quad

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occupancy between the 4 drug/diet groups. There was also no difference in the mean proximity to the former location of the escape platform (data not shown), suggesting no significant effect of either diet

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or drug on spatial memory under these experimental conditions. In summary, mice with either CGP 39551 and/or aspartame exposure exhibited impaired spatial

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navigation in the MWM paradigm. Thigmotactic and passive floating, behaviors associated with anxiety and passivity, was evident in both aspartame-fed groups of mice.

3.4.

Principal component analysis (PCA).

The results of the analysis indicated that specific variables of the different behavioral tests were differentially affected by diet and NMDAR antagonism. To extract a more significant underlying factor across tests, dimensional reduction analysis using PCA was performed. The reliability of PCA depends on the variables used, and it is important to avoid extreme multicollinearity which are commonly detected

by

Pearson

correlation

analysis.

Accordingly,

those

variables which

contributed

to multicollinearity and correlated >0.70 were excluded. On this basis, only the aforementioned variables on Day 3 were retained for the final analysis. PCA of selected variables from all tests were extracted into three factors using the scree-plot, which accounted for 71.09% of total variance. The three factors did not correlate with each other and the variables with low communalities (<0.4) were

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ACCEPTED MANUSCRIPT not included in the analysis. Varimax rotation with Kaiser Normalization was used to extract and load all the variables accordingly and is shown in Table 4. The three factors were: a. Factor 1: Floating on Day 3 of MWM, mean velocity, total mobility, total distance, sectors crossed,

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center duration and frequency, and periphery frequency in OF are the variables that loaded positively, whereas Path length on Day 3 of MWM, contracted posture and periphery duration in

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OF loaded negatively. This factor accounted for 46.05% of the variance and was termed

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“locomotor activity and anxiety”.

b. Factor 2: The variables that loaded positively on this factor were latency to platform, path length, thigmotaxis on Day 3 of MWM and STM total exploration time in Object Recognition Test.

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However, the time spent on platform and speed on Day 3 of MWM loaded negatively. This factor accounted for 16.78% of the variance and was termed “spatial navigation and exploration”.

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c. Factor 3: The variables that loaded positively on this factor were floating on Day 3 of MWM, periphery frequency in OF, total exploration time in training phase and LTM. It contributed to

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9.08% of the variance and this factor was termed “risk assessment and memory impairment”. The results of the PCA were taken into consideration during the final assignation of phenotypes to

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describe behavioral characteristics of aspartame-exposed mice. The fact that the independent behavioral variables differentially affect these behaviors suggests that they represent diverse

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strategies involved in a complex behavioral response to anxiogenic situations. The first factor (‘floating and locomotor activity’) reveals the differences in locomotor activity between the diet and CGP 39551 drug-treated mice, with ASP+CGP mice exhibiting a hypolocomotor trait not seen in the other groups. We also observed floating and increased peripheral occupation in ASP+CGP group mice, which could be attributed to an effect of independent variables on Factor 3, “anxiety and risk assessment.”

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ACCEPTED MANUSCRIPT 4.

Discussion

The main outcomes of the present study are that lifetime exposure to aspartame commencing in utero via the mother’s diet may elevate blood glucose and impair insulin-stimulated glucose disposal during an

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ITT; and that this can be normalized by early-life intervention with the competitive NMDAR antagonist drug CGP 39551. Since these effects were not apparent in control diet mice with NMDAR antagonism,

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this suggests a drug/diet effect early in life. Additionally, early-life NMDAR antagonism led to diet-

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specific modulations in adulthood behavioral phenotype. Essentially, NMDAR antagonism in control diet mice resulted in hyperlocomotion, whereas aspartame diet mice with NMDAR antagonism were

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hypolocomotive and displayed anxiogenic-type behavior. Mechanisms for the differential effects observed are likely to involve drug/diet interactions leading to modulation of neuroendocrine pathways

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involved in energy homeostasis and behavior, since if CGP 39551 exerted its effect via epigenetic modulations unrelated to NMDA receptor signaling, it would be expected that both Control diet and aspartame-exposed mice would have responded to NMDAR antagonism in an identical manner.

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The central nervous system (CNS) is pivotal in regulating glucose homeostasis and the

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counter regulatory response (CRR) to hypoglycemia [24,45]. Under normal conditions, information from the activity of afferent neurons together with postprandial changes in nutrient and hormone levels are

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relayed to the CNS which then responds by increasing autonomic efferent activity to direct changes in hepatic gluconeogenesis and glucose uptake by peripheral tissues [46]. The hypothalamus contains several types of glucose-sensing neurons which respond to changes in extracellular glucose concentration by altering their firing rate [47]. Recent work has highlighted the importance of NMDARmediated glutamatergic signaling and synaptic plasticity in the sympathoexcitatory control of AgRP neuronal activity [48], which can be potentiated by insulin [49]. Projections from glucose-inhibited AgRP neurons in the arcuate (ARC) nucleus activate orexin/hypocretin neurons in the lateral hypothalamus [47] in order to control glucose production and utilization in the peripheral tissues via activation of the autonomic nervous system [50] which is also subject to glutamatergic control. NMDA receptor agonists have been shown to increase orexin activity, whereas NMDAR antagonists and GABAergic input reduces it [51,52]. Similarly, projections from glucose-excited ARC POMC neurons activate melanin concentrating hormone (MCR) neurons in the lateral hypothalamus to regulate glucose homeostasis and behavior [53] The insulin tolerance test (ITT) is considered an appropriate method of assessing insulin sensitivity, which is impaired in diabetics [54]; and the normal response to exogenous insulin challenge 16

ACCEPTED MANUSCRIPT is a rapid and transient reduction in blood glucose levels which is followed by activation of various neuroendocrine pathways involved in the CRR to insulin-induced hypoglycemia. The ITT is also used in a clinical setting to assess the integrity of the hypothalamic pituitary adrenal axis [55], which is involved in

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glycemic control during hypoglycemia [47] and which is sensitive to NMDAR modulation [56]. The mechanism whereby aspartame ingestion affects glucose disappearance during the ITT is still under however

aspartame-induced

dysbiosis

[26,27]

could

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investigation,

potentially

alter

CNS

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neurotransmitters via modulation of the microbiota-gut-brain axis [57,58]. In particular serotonin, a neurotransmitter active at both terminals of the microbiota-gut-brain axis [58], has been shown to be altered in the CNS following aspartame exposure [59]. It has long been known that serotonergic

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mechanisms can moderate the neuroendocrine responses to insulin, and both orexin and serotonin have recently been shown to modulate the epinephrinergic CRR response in the perifornical

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hypothalamus [60]. In our study, NMDAR antagonism attenuated the aspartame-induced glucose impairment during the ITT. Previously, infusions of the NMDAR antagonist MK-801 during insulin

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challenge has also been shown to result in a more rapid attenuation in plasma glucose levels [45]; however we did not find a similar improvement in leaner control diet mice treated with CGP in which

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the insulin sensitivity was comparable with untreated controls. It is therefore possible that CGP 39551 and aspartame treatment may have differentially affected mechanisms in the HPA axis which are

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involved in the CRR to insulin challenge during the ITT. Interestingly, functional NMDARs have been recently characterized in pancreatic beta cells [61,62]. The non-competitive NMDAR antagonist dextromethorphan has been demonstrated to enhance glucose-stimulated insulin secretion (GSIS) and survival of islet cells in vitro, and enhance glucose tolerance in vivo (61), prompting considerable interest in NMDAR antagonistic pharma as an adjunct treatment for diabetes in the future. Additionally, whereas MK-801 did not affect GSIS in the pancreatic clonal BRIN-BD11 beta cell line, the NMDAR modulator homocysteine suppressed basal and GSIS; and the combination of homocysteine and MK-801 further impaired GSIS, providing further evidence for a role of NMDARs in pancreatic beta cell function [62]. Moreover, in the same way that hypothalamic control of glucose and energy homeostasis is vulnerable to metabolic imprinting in utero [19,20], pancreatic beta cell function may be affected by environmental factors such as maternal diet and gut microbiota [63], which could alter NMDAR sensitivity. Taken together, these studies highlight the importance of NMDARs in the regulation of energy balance and glycemic control; and provide insight 17

ACCEPTED MANUSCRIPT into the mechanism(s) behind the modulation of aspartame-induced glucose deregulation by NMDAR antagonism seen in the present study.

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Aspartame ingestion has also been demonstrated to result in a variety of behavioral impairments in rodents [11,13,64,65] and in some [66-69], but not all [70] human studies. In the present

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study ASP diet mice retained similar characteristics to control mice in terms of locomotor activity, but exhibited impaired spatial navigation and place learning in the MWM paradigm. Several possibilities

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exist for the mechanism(s) involved, including aspartame-induced dysbiosis [26,27] which could potentially alter CNS neurotransmitters, serotonin in particular [71], via modulation of the microbiota-

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gut-brain axis [57,58]. Changes in both the gut microbiota and the serotinergic system occur across the lifespan and have substantial periods of developmental overlap during which a reciprocal influence is

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possible [58], and during which NMDAR antagonism could further modulate behavior in an aspartamespecific manner.

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In agreement with previous studies [33,72], we found that NMDAR antagonism in control diet

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mice caused adulthood hyperlocomotion and spatial navigation impairment during the MWM test. In contrast, whereas aspartame diet mice exhibited impaired spatial cognition and anxiogenic behavior

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without overt locomotor defects; aspartame-exposed mice with NMDAR antagonism exhibited hypolocomotion, reduced exploratory and increased anxiety-related behavior together with spatial navigation defects. Additionally serum corticosterone levels were raised in pair-fed ASP+CGP mice that did not undergo behavioral testing, suggesting a direct effect of the drug/diet interaction independent of behavioral testing which is known to raise corticosterone levels [42]. Interestingly, in addition to regulating glucose homeostasis, both hypothalamic NMDAR-expressing AgRP and POMC type neurons can modulate locomotion and stress-linked behavior [73-75], which is associated with the hypothalamic pituitary adrenal (HPA) axis. In addition to their aforementioned effects on glucose homeostasis, projections from AgRP neurons in the arcuate nucleus activate orexin/hypocretin neurons in the lateral hypothalamus to increase locomotor activity, and to regulate sleep/wakefulness states, exploratory behavior and stress responses [47]. Conversely, melanin concentrating hormone (MCH)-neurons in the lateral hypothalamus are activated by POMC neurons projecting from the arcuate nucleus to promote sleep and energy conservation [47].

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ACCEPTED MANUSCRIPT Our experiments demonstrated largely beneficial effects of CGP 39551 on insulin tolerance in aspartame-fed mice, whereas differential effects on locomotion, anxiogenic behavior and spatial navigation were observed. Several hypotheses may be proposed to explain this functional diversity.

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Firstly, it is likely that different first- and second-order neuronal networks are involved in the regulation of the physiological and behavioral parameters under investigation, and these may express different

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combinations of NMDA receptor subunits which are known to be spatiotemporally regulated to coincide

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with critical stages in development [76]. Thus, neurons expressing heterogeneous levels of receptor subunits are likely to be differentially affected by maternal CGP 39551 administration [77], in addition to regional and temporal differences in the concentration of agonists, as well as co-agonists such as glycine

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and its binding-site competitor phenylalanine [78]. It follows that the differential effects of CGP 39551 on physiological and behavioral parameters in aspartame-exposed mice may be related to the

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developmental time-point at which these various processes occur and are therefore more vulnerable to NMDAR antagonism. Whereas long-term programming of glucose homeostasis [79], regulation of the

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HPA axis and anxiety-type behavior [80] may occur in utero during hypothalamic development; some of the neuronal networks involved in governing spatial learning and cognition only emerge during the first

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during this time.

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3 weeks of rodent life [81], and may have been differentially modulated by the drug/diet administration

In conclusion, we have developed a model in which early-life administration of the competitive NMDAR antagonist drug CGP 39551 may attenuate aspartame-induced impairment in adulthood insulin tolerance. Secondly, the drug intervention led to diet-specific modulations in adulthood behavioral phenotype, further suggestive of a drug/diet interaction occurring early in life. These findings suggests a possible involvement of early specific NMDAR interactions in aspartameimpaired glucose homeostasis and behavioral deficits.

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ACCEPTED MANUSCRIPT Acknowledgments: We are indebted to Drs. Abdullah Assiri and Falah Almohanna from the Comparative Medicine Department, KFSH&RC for their invaluable advice and cooperation in conducting the animal

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experiments; and L. Al Mohanna for many helpful discussions. Funding: This study was funded by a grant from the National Science and Technology Innovation

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Program (Grant# MED-13-2506-20).

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ACCEPTED MANUSCRIPT Figure and Table Captions.

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Figure 1: Schematic of experimental design. (A) overview (B) timeline. Table 1: Effect of diet and early-life CGP 39551 administration on food and fluid intake together with

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body characteristics.

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Figure 2: Early-life NMDAR antagonism attenuates aspartame-induced insulin intolerance. (A) Effect of diet on glucose levels during a random fed insulin tolerance test. Effect of CGP 39551 on Control diet

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mice (B), and Aspartame diet mice (C), Area Under the Curve analysis of glucose levels (D). Data are presented as Means ± SEM, N=18 per group. A significance of P-value <.05, <0.01 and <0.001 based on independent t-tests are indicated by *, **and *** for CON vs all other groups; $$ for ASP vs ASP+CGP

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when p<0.01.

Table 2: Effect of diet and early-life CGP 39551 intervention on glucose parameters during a random-fed

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insulin tolerance test.

Table 3: Effect of diet and NMDAR antagonism on behavioral parameters during the Open Field test.

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Figure 3: Effect of diet and CGP 39551 administration on (A) Behavior in the Dark/light transition test. (B) Total exploration time and (C) Object Discrimination Index during an Object Recognition Test. Data are presented as Means ± SEM, N=18 per group. A significance of P-value <.05 and <0.01 based on independent t-tests are indicated by * and ** for CON vs all other groups; $ for ASP vs ASP+CGP when p<0.05.

Figure 4: Effect of diet and NMDAR antagonism on behavioral parameters during the MWM test. (A) Latency to reach the platform and (B) Area Under the Curve analysis of escape latency during the 4 days of acquisition training. (C) Time spent in location of hidden platform and (D) Area Under the Curve analysis of time spent in location of the hidden platform during the 4 days of acquisition training. Data are presented as Means ± SEM, N=18 per group. A significance of P-value <.05, <.01 and <0.001 based on independent t-tests are indicated by *, ** and *** for CON vs all other groups and $ for ASP vs ASP+CGP when p<0.05.

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ACCEPTED MANUSCRIPT Figure 5: Effect of diet and CGP 39551 administration on behavioral parameters during the MWM test. (A) Thigmotactic behavior (B) Area Under the Curve analysis of thigmotactic behavior during the 4 days of acquisition training. (C) Time spent in directionless floating and (D) Area Under the Curve analysis of

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time spent in directionless floating during the 4 days of acquisition training. Data are presented as Means ± SEM, N=18 per group. A significance of P-value <.05 and <.01 based on independent t-tests are

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indicated by * and **for CON vs all other groups and † for CON + CGP vs ASP+CGP when p<0.05.

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Table 4: Factors and factor loading obtained by PCA of selected variables in aspartame-fed mice.

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43. Vasuta C, Caunt C, James R, Samadi S, Schibuk E, Kannangara T, Titterness AK, Christie BR. Effects of exercise on NMDA receptor subunit contributions to bidirectional synaptic plasticity in the mouse dentate gyrus. Hippocampus. 2007; 17(12): 1201-8. 44. Kaneko JJ, Harvey JW, Bruss ML. Clinical biochemistry of domestic animals. New York Academic Press; 1999. 45. Molina PE, Abumrad NN. Contribution of excitatory amino acids to hypoglycemic counterregulation. Brain Res. 2001; 899(1-2) :201-8. 46. Sisley S, Sandoval D. Hypothalamic control of energy and glucose metabolism. Rev Endocr Metab Disord. 2011; 12(3): 219-33. 47. Burdakov D, Luckman SM, Verkhratsky A. Glucose-sensing neurons of the hypothalamus. Philos Trans R Soc Lond B Biol Sci. 2005; 360(1464): 2227-35. 48. Liu T, Kong D, Shah BP, Ye C, Koda S, Saunders A, Ding JB, Yang Z, Sabatini BL, Lowell BB. Fasting activation of AgRP neurons requires NMDA receptors and involves spinogenesis and increased excitatory tone. Neuron. 2012; 73(3): 511-22 49. Skeberdis VA, Lan J, Zheng X, Zukin RS, Bennett MV. Insulin promotes rapid delivery of N-methylD- aspartate receptors to the cell surface by exocytosis. Proc Natl Acad Sci U S A. 2001; 98(6): 3561-6. 50. Tsuneki H, Wada T, Sasaoka T. Role of orexin in the regulation of glucose homeostasis. Acta Physiol (Oxf). 2010; 198(3):335-48 51. Li Y, Gao XB, Sakurai T, van den Pol AN. Hypocretin/Orexin excites hypocretin neurons via a local glutamate neuron-A potential mechanism for orchestrating the hypothalamic arousal system. Neuron. 2002; 36(6):1169-81. 52. Yamanaka A, Muraki Y, Tsujino N, Goto K, Sakurai T. Regulation of orexin neurons by the monoaminergic and cholinergic systems. Biochem Biophys Res Commun. 2003; 303(1):120-9. 53. Kosse C, Gonzalez A, Burdakov D. Predictive models of glucose control: roles for glucose-sensing neurones. Acta Physiol (Oxf). 2015; 213(1):7-18. 54. Kozawa J, Iwahashi H, Okita K, Okauchi Y, Imagawa A, Shimomura I. Insulin tolerance test predicts the effectiveness of insulin sensitizers in Japanese type 2 diabetic patients. Diabetes Ther. 2010; 1(2): 121-30. 55. Ajala O, Lockett H, Twine G, Flanagan DE. Depth and duration of hypoglycaemia achieved during the insulin tolerance test. Eur J Endocrinol. 2012; 167(1): 59-65. 56. Kent S, Kernahan SD, Levine S. Effects of excitatory amino acids on the hypothalamic-pituitaryadrenal axis of the neonatal rat. Brain Res Dev Brain Res. 1996; 94(1): 1-13. 57. Cryan JF, Dinan TG. Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nat Rev Neurosci. 2012; 13(10):701-12. 58. O'Mahony SM, Clarke G, Borre YE, Dinan TG, Cryan JF. Serotonin, tryptophan metabolism and the brain-gut-microbiome axis. Behav Brain Res. 2015; 277:32-48. 59. Sharma RP, Coulombe RA Jr. Effects of repeated doses of aspartame on serotonin and its metabolite in various regions of the mouse brain. Food Chem Toxicol. 1987; 25(8): 565-8.

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60. Otlivanchik O, Sanders NM, Dunn-Meynell A, Levin BE. Orexin signaling is necessary for hypoglycemia-induced prevention of conditioned place preference. Am J Physiol Regul Integr Comp Physiol. 2016; 310(1):R66-73. 61. Marquard J, Otter S, Welters A, Stirban A, Fischer A, Eglinger J, Herebian D, Kletke O, Klemen MS, Stožer A, Wnendt S, Piemonti L, Köhler M, Ferrer J, Thorens B, Schliess F, Rupnik MS, Heise T, Berggren PO, Klöcker N, Meissner T, Mayatepek E, Eberhard D, Kragl M, Lammert E. Characterization of pancreatic NMDA receptors as possible drug targets for diabetes treatment. Nat Med. 2015; 21(4): 363-72. 62. Patterson S, Irwin N, Guo-Parke H, Moffett RC, Scullion SM, Flatt PR, McClenaghan NH. Evaluation of the role of N-methyl-D-aspartate (NMDA) receptors in insulin secreting beta-cells. Eur J Pharmacol. 2016; 771: 107-13. 63. Nielsen JH, Haase TN, Jaksch C, Nalla A, Søstrup B, Nalla AA, Larsen L, Rasmussen M, Dalgaard LT, Gaarn LW, Thams P, Kofod H, Billestrup N. Impact of fetal and neonatal environment on beta cell function and development of diabetes. Acta Obstet Gynecol Scand. 2014; 93(11): 1109-22. 64. Christian B, McConnaughey K, Bethea E, Brantley S, Coffey A, Hammond L, Harrell S, Metcalf K, Muehlenbein D, Spruill W, Brinson L, McConnaughey M. Chronic aspartame affects T-maze performance, brain cholinergic receptors and Na+, K+-ATPase in rats. Pharmacol Biochem Behav. 2004; 78(1): 121-7. 65. Abdel-Salam OM, Salem NA, El-Shamarka ME, Hussein JS, Ahmed NA, El-Nagar ME. Studies on the effects of aspartame on memory and oxidative stress in brain of mice. Eur Rev Med Pharmacol Sci. 2012; 16(15):2092-101. 66. Konen JA, Sia,TL, Czuchry M, Stunz PM, Bahr GS, Barth TM, Dansereau DF. Perceived memory impairment in aspartame users. Presented at the Society for Neuroscience 30th Annual meeting, 2000; New Orleans, LA. 67. Sünram-Lea SI, Foster JK, Durlach P, Perez C. Investigation into the significance of task difficulty and divided allocation of resources on the glucose memory facilitation effect. Psychopharmacology (Berl) 2002; 160(4): 387-97. 68. Reid M, Hammersley R, Hill AJ, Skidmore P. Long-term dietary compensation for added sugar: effects of supplementary sucrose drinks over a 4-week period. Br J Nutr. 2007; 97(1): 193-203. 69. Lindseth GN, Coolahan SE, Petros TV, Lindseth PD. Neurobehavioral effects of aspartame consumption. Res Nurs Health 2014; 37(3): 185-93. 70. Spiers PA, Sabounjian L, Reiner A, Myers DK, Wurtman J, Schomer DL. Aspartame: neuropsychologic and neurophysiologic evaluation of acute and chronic effects. Am J Clin Nutr 1998; 68(3): 531-7. 71. Humphries P, Pretorius E, Naude H. Direct and indirect cellular effects of aspartame on the brain. Eur J Clin Nutrition 2008; 62: 451-462. 72. Bubeníková-Valesová V, Horácek J, Vrajová M, Höschl C. Models of schizophrenia in human and animals based on inhibition of NMDA receptors. Neurosci Behav Rev 2008: 32: 1014-1023

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73. Huang H, Lee SH, Ye C, Lima IS, Oh BC, Lowell BB, Zabolotny JM, Kim YB. ROCK1 in AgRP neurons regulates energy expenditure and locomotor activity in male mice. Endocrinology. 2013; 154(10): 3660-70. 74. Mesaros A, Koralov SB, Rother E, Wunderlich FT, Ernst MB, Barsh GS, Rajewsky K, Brüning JC. Activation of Stat3 signaling in AgRP neurons promotes locomotor activity. Cell Metab. 2008; 7(3): 236-48. 75. Greenman Y, Kuperman Y, Drori Y, Asa SL, Navon I, Forkosh O, Gil S, Stern N, Chen A. Postnatal ablation of POMC neurons induces an obese phenotype characterized by decreased food intake and enhanced anxiety-like behavior. Mol Endocrinol. 2013; 27(7): 1091-102. 76. Low CM, Wee KS. New insights into the not-so-new NR3 subunits of N-methyl-D-aspartate receptor: localization, structure, and function. Mol Pharmacol. 2010; 78(1): 1-11. 77. Blaise MC, Sowdhamini R, Pradhan N. Comparative analysis of different competitive antagonists interaction with NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) ionotropic glutamate receptor. J Mol Model. 2005; 11(6): 489-502. 78. Glushakov AV, Dennis DM, Sumners C, Seubert CN, Martynyuk AE. L-phenylalanine selectively depresses currents at glutamatergic excitatory synapses. J Neurosci Res. 2003; 72(1): 116-24. 79. Steculorum SM, Vogt MC, Brüning JC. Perinatal programming of metabolic diseases: role of insulin in the development of hypothalamic neurocircuits. Endocrinol Metab Clin North Am. 2013; 42(1): 149-64. 80. Seckl JR. Prenatal glucocorticoids and long-term programming. Eur J Endocrinol. 2004; 151 Suppl 3:U49-62. 81. Wills TJ, Muessig L, Cacucci F. The development of spatial behaviour and the hippocampal neural representation of space. Philos Trans R Soc Lond B Biol Sci. 2013; 369(1635): 20130409

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ACCEPTED MANUSCRIPT Table 1 Effect of diet and early-life CGP 39551 administration on food and fluid intake together with body characteristics. CON

CON + CGP

ASP

ASP + CGP

28.61 ± 0.45

29.41 ± 0.35

31.06 ± 0.31 4.69 ± 0.25 4.14 ± 0.15 45.44 ± 1.16

Pvalue Diet*Drug

Interscapular fat (g)

0.088 ± 0.00

0.094 ± 0.01

0.235 ± 0.01

s

†

0.252 ± 0.01

0.253 ± 0.01 †

Glucose (mg/dL)

135.58 ± 4.6

138.14 ± 3.17

Plasma Insulin (ulU/ml) Corticosterone (ng/ml)

7.61 ± 0.09 161.6 ± 11.07

8.67 ± 0.73 185.79 ± 14.07

± SEM, n=18 per group.

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Data presented are Means

0.662 ± 0.04 0.099 ± 0.01

SC

Pancreas (g)

††

0.528 ± 0.05

0.007

4.85 ± 0.43 4.06 ± 0.09 46.59 ± 0.68

0.318 0.06

0.457$$ ± 0.04

0.005

†$

0.002

PT

3.91 ± 0.23 3.86 ± 0.13

29.72$$ ± 0.36

RI

Food Intake (g/day) 4.37 ± 0.31 Fluid intake (ml/day) 3.72 ± 0.12 Aspartame intake (mg/Kg BW) White adipose tissue (g) 0.500 ± 0.03

††

148.56 ± 4.55 12.88 ± 4.78 152.89 ± 14.70

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Final weight (g)

0.087 ± 0.01 0.258 ± 0.01 $

0.55

135.06 ± 4.28

0.01

7.74 ± 0.13 193.35$ ± 12.06

0.204 0.418

P-values for Diet* Drug effect were calculated using 2-way A NOVA,

†

A significance of P-value <.05 and <0.01 based on independent t-tests are indicated by and

††

for significance between diet groups (CON vs ASP;

D

CON+CGP vs ASP+CGP); and significant drug effect within each diet (i.e. CON vs CON+CGP and ASP vs ASP+CGP) are represented using $ and $$ respectively.

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CON: control; CON -J-CGP: control with CGP 39551; ASP: Aspartame; ASP -i-CGP: Aspartame with CGP 39551; BW: Body weight.

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ACCEPTED MANUSCRIPT Table 2 Effect of diet and early-life CGP 39551 intervention on glucose parameters during a random-fed insulin tolerance test. ASP

ASP + CGP

6291 ± 176

6788 ±173

7652 299

Kitt (%/m in)

1.56 ± 0.12

1.28 ± 0.10

0.94

46.14 ± 3.14

67.65

^1/2 GLUCOSE tm i n)

††

±

††

6557

†

67.7 ± 7.74

0.001 0.342

52.59 ± 4.07

0.403

SC

Data presented are Means! SEM, n=18 per group.

± 231

Sig.

± 0.14

1.2

±0.12 ± 7.47

$

PT

CON + CGP

RI

CON AuCglucose {mg/dL.min)

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P-values for Diet*Drug effect were calculated using 2-way ANOVA.

MA

A significance of P-value <.05 and <0.01 based on independent t-tests are indicated by † and †† for significance between diet groups (CON vs ASP; CON+CGP vs ASP+CGP); and significant drug effect within each diet (i.e. CON vs CON+CGP and ASP vs ASP+CGP) are represented using $ and $$ respectively.

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TE

D

CON: control; CON + CGP: control with CGP 39551; ASP: Aspartame; ASP + CGP: Aspartame with CGP 39551

35

ACCEPTED MANUSCRIPT Table 3 Effect of diet and NMDAR antagonism on behavioral parameters during the Open Field test. p-value CON

CON + CGP

ASP

ASP + CGP diet*drug

Maximum velocity (m/s) Mean velocity (m/s)

38.85 ± 0.9 8.88 ± 0.28

43.21 ± 2.26 9.33 ± 0.55

38.9 ± 1.21 8.79 ± 0.46

Immobile duration (s)

80.85 ± 4.47

75.01 ± 4.94

82.09 ± 6.99

190.05 ± 3.92

Highly mobile duration (s) 28.2 ± 1.59

0.008 0.013

††$

± 8.13

0.013

± 6.06

0.116

± 2.81

0.002

± 8.13

0.013

± 113

0.013

13.01 ± 2.69 200.94 ± 6.32

0.053

108.6

186.92 ± 3.5

186.99 ± 4.29

41.5$ ± 6.05

30.91 ± 4.11

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Mobile duration (s)

††

35.88 ± 1.18 †† 7.07 $$ ± 0.38

PT

$

219.15 ± 4.47

224.99 ± 4.94

217.91 ± 6.99

Total distance (cm)

2662 ± 83

2800 ± 163

2638 ± 137

SC

Total mobility (s)

5.98 ± 0.81

6.92 ± 1.01

5.71 ± 1.22

Normal posture (s) Stretched posture (s)

204.61 ± 4.03 89.45 ± 4.17

195.9 ± 4.77 97.21$ ± 5.4

192.52 ± 6.74 101.81 ± 7.7

Rearing frequency

10.94 ± 2.04

14.11 ± 1.88

NU

Contracted posture (s)

8.00 ± 1.4

18.84 191.4 2120

8.17

3.11 ± 0.43 294.17 ± 18.14

4.82$ ± 1.11 270.56 ± 15.81

Centre zone frequency

8.83 ± 1.03

9.22 ± 1.24

6.67 ± 0.89

Centre zone duration (s)

9.83 t 1.3

8.34 ± 1.51

7.08 ± 0.94

3.79

Periphery zone frequency

25.11 ± 1.44

25.06 ± 2.32

21.94 ± 1.49

16.5

D

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39.72 ± 2.19

2.71 ± 0.41 276.72 t 9.31

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262.55 ± 3.14

†$

††

± 6.31

29.83 ± 2.75 2.19 ± 0.36 212.72 3.72

††$$

††$ †$$

0.129 0.003 0.122

± 1.78

††$

36.5 ± 2.02

254.78 ± 4.21

††$

††$$

86.08

Grooming frequency Total sectors crossed

†

††$

†$

Total Vertical Activity

Periphery zone duration(s) 250.82 ± 3.78

34.06 ± 2.03

168.76

†$

± 13.8

0.021 0.024 0.012

± 0.65

0.092

± 0.74

0.446

± 1.64

0.130

††$

0.434

††$

272.31

± 3.52

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Data presented are Means ± SEM, n=18 per group. P-values for Diet*Drug effect were calculated using 2-way ANOVA. †

††

A significance of P-value <.05 and <0.01 based on independent t-tests are indicated by and for significance between diet groups (CON vs ASP; CON+CGP vs ASP+CGP); and significant drug effect within each diet (i.e. CON vs CON+CGP and ASP vs ASP+CGP) are represented using $ and $$ respectively. CON: control; CON + CGP: control with CGP 39551; ASP: Aspartame; ASP

+ CGP: Aspartame with CGP 39551; BW: Body weight

36

ACCEPTED MANUSCRIPT Table 4 Factors and factor loading obtained by PCA of selected variables in aspartame-fed mice. Variables

Factors 2

-.540

.362 .647 -.552

.407

Thigmotime Day 3 - MWM Time spent in platform Day 3 - MWM

Contracted posture - OF

-.316

Total distance covered - OF

.913

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.913 .923

Total sectors crossed - OF

.927 .676

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Centre frequency - OF

Total exploration time - Training Total exploration time - STM

.504

.551 .751

Mean velocity - OF Total mobility - OF

Periphery duration - OF Periphery frequency - OF

-.569

SC

Floating Day 3 - MWM

RI

Speed Day 3-MWM

3

PT

Latency Day 3 - MWM Path Day 3 - MWM

1

.895 -.830 .810

.418 .707 .636

Total exploration time - LTM

.779

Extraction Method: Principal Component Analysis. Rotation Method: Van max with Kaiser Normalization Loadings >0.4 only shown

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ACCEPTED MANUSCRIPT

Highlights: Effects of maternal aspartame consumption on offspring physiology and behavior were studied.



Chronic aspartame exposure commencing in utero impaired insulin tolerance.



Prenatal NMDA receptor antagonism improved aspartame-impaired insulin tolerance.



Prenatal NMDA receptor antagonism caused diet-specific modulations in offspring behavior.



Data suggests the involvement of NMDA receptors in aspartame-induced effects on physiology

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and behavior.

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