1 CHAPTER ONE 1.0 INTRODUCTION Aspartame (L

CHAPTER ONE 1.0

INTRODUCTION Aspartame (L-phenylalanine N-L-·-aspartyl-1-methyl ester) is one of the most widely used

synthetic sweeteners available (Maher and Wurtman, 1987); a methyl-ester of a dipeptide made up of aspartate and phenylalanine (Plate 1), it is approved for use in over a 100 countries (Aspartame information centre, 2006). Aspartame is a white crystalline, odourless but intensely sweet powder sold under the brand names NutraSweet® and Equal®; believed to be about 200 times sweeter than sucrose (Magnuson et al., 2007). The metabolism of aspartame provides approximately 4 kcal/g of energy (Gougeon et al., 2004). This energy, although similar to the caloric value of sucrose is negligible as the high-intensity sweetening power of aspartame means that little is needed to be added to foods to achieve sweetness (Magnuson et al., 2007). Aspartame was discovered by James Schlatter of the G.D. Searle Company in 1965 (Garriga and Metcalfe, 1988). Schlatter, was at the time a scientist in the G. D. Searle research laboratory working on the synthesis of a gastrin inhibitor, he was attempting to crystallize aspartyl phenylalanine methyl-ester when some of the liquid spilled on his hands, he licked it inadvertently and realised the intense sweetness of the chemical (Manzur, 1984). It was approved for consumption in 1981, 17 years after by the United States Food and Drug Administration (FDA) (Stegink, 1987), and in 1994, 29 years after by the European Union as published by the Brazilian food safety agency (ABIAD) (ABIAD, 1994). These periods were marked by controversy over its innocuousness leading to requests for toxicological studies that still persists to date (Aspartame information centre, 2006). Reports by the French food safety agency (AFFSA) revealed that aspartame can be found in a wide variety of pharmaceutical products and is also approved for use in well over 6000 food products (AFSSA, 2002), amongst its consumers are people trying to lose weight or people with diabetes, including children, who are looking for unsweetened or sugar-free products. Over 200 million people consume aspartame worldwide (Shapiro 1988). 1

Plate 1: Structure of aspartame (N-l-alpha-aspartyl-l-phenylalanine 1-methylester). Adapted from Alsuhaibani et al., 2010.

2

The use of aspartame by diabetics continues to increase (Shapiro 1988) and a recent observation has revealed that aspartame is slowly making its way into every day products, which do not carry any indication of being for people on diet or diabetics. Thus, aspartame is used not only by the above-mentioned group of people, but also by unsuspecting individuals (Humphries et al., 2008). Available data has shown that over two thousand tons of aspartame per year is consumed by 375 million people in Europe (Aspartame information service, 2006), while a study carried out on the American population between 1984 and 1992, showed that the average daily intake of aspartame is 2-3 mg/kg body weight in adults and 2.5-5 mg/kg body weight in children because of the lower body weight (Butchko et al., 2002), though these amounts are far from the recommended maximum daily intake, the rapidly-widening arsenal of foods containing aspartame means that the recommended maximum daily intake {40 mg/kg body weight in Europe and 50 mg/kg body weight in the United States (Butchko et al., 2002)} is approachable by simultaneous consumption of food products containing aspartame. There is increasing concern on possible adverse neurological and behavioural effects of aspartame or its metabolic components (phenylalanine, aspartic acid (aspartate), diketopiperazine and methanol), which are produced during its breakdown. There have been reports on neurochemical effects aspartame consumption (Goerss et al., 2000); some researchers regardless are of the opinion that aspartame is safe (Butcko et al., 2002). This divergence of research opinion on the effects of aspartame is still continuing 30 years after the FDA had approved its use. The controversies surrounding aspartame’s safety continue to date with different benefactors of the contending opinions funding research and safety reviews or publishing blogs to further buttress their point. The unsuspecting consumer lies in the thick of it all not knowing which school of thought to buy into. The effects of aspartame have been studied in various species, including humans, rats, mice and rabbits. Most studies described in the literature have a macroscopic approach. If no adverse effects are visible after a single large administered dose of aspartame, it is believed that aspartame has no 3

effect (Humphries et al., 2008). Results obtained from different studies vary from severe adverse effects to none; further studies need to be carried out microscopically to demonstrate possible adverse effects on the cellular level that may not be obvious macroscopically. 1.1

OBJECTIVES OF STUDY: The specific objectives are to: (a) determine the effects of aspartame on the histomorphology of the cerebral cortex, cerebellum and hippocampus, (b) determine the effect of aspartame on neuritic plaque formation in the cerebral cortex and hippocampus, (c) assess the effects of aspartame on brain neurogenic markers; Glial Fibrillary Acidic Protein (GFAP) and Neuron Specific Enolase (NSE), (d) assess the excitatory or inhibitory effect of oral aspartame using the Open field; and (e) determine the effects of aspartame on anxiety and memory using the Elevated plus maze and Y maze.

1.2

STATEMENT OF RESEARCH PROBLEM Aspartame is one of the most widely consumed artificial food sweeteners; while there is

increasing concerns on possible adverse neurological and neurobehavioural effects following its consumption, there is a divergence of research opinion on these effects. This research seeks to investigate the effects of aspartame on the histomorphology of the cerebral cortex, hippocampus and cerebellum; neurogenesis and neurobehaviour in mice.

1.3

EXPECTED CONTRIBUTION TO KNOWLEDGE The results of the study will provide information on the effects of aspartame on the brain

of adult male Swiss mice, especially in relation to behavioural and morphological changes associated with the cerebral cortex, hippocampus and cerebellar cortex. 4

CHAPTER TWO 2.0

LITERATURE REVIEW

2.1

ASPARTAME-CONTAINING CONSUMER PRODUCTS Aspartame has become an important component in many low-calorie, sugar-free foods and

beverages and this is largely responsible for its growth over the last two decades in the sugar-free market. Aspartame is consumed by over 200 million people worldwide and is found in over 6,000 products amongst which are carbonated soft drinks (diet Coke, diet Pepsi, Coca cola zero, 7 Up) powdered soft drinks (Tango, Cola, Nutri C) chewing gum (Orbit, Wrigleys), confections, gelatins, yogurt, tabletop sweeteners, condiments (Ketchup, salad dressings, barbeque sauce) and some pharmaceuticals such as vitamins and sugar-free cough drops (Aspartame information centre, 2015).

2.2

ESTIMATED DAILY CONSUMPTION OF ASPARTAME Aspartame does not occur naturally in foods, estimates of its consumption are largely

based on amounts intentionally added to food. The consumption of aspartame has been studied in the general population and in special population subgroups; this is done by calculating food intake and multiplying that by the amount of aspartame in these food (Magnuson et al., 2007). Estimates of projected maximum consumption of aspartame calculated by the FDA, the Market Research Corporation of America, and various researchers in the 1970s ranged from 22 to 34 mg/kg body weight/day (Stegink, 1987). Studies in New Zealand and Australia by the food standards Australia and New Zealand (FSANZ) assessed the average daily consumption of aspartame amongst individuals, doing so by breaking them down into age brackets: 12 to 17, 18 to 24, 25 to 39, 40 to 59, and over 60 years. Highest consumption was seen in individuals aged between 25 and 39 with a mean of 3.4 mg/kg body weight/day and 95th percentile value of 9.89 mg/kg body weight/day. Children aged between 12 and 17 consumed approximately half of these values (mean, 1.75 mg/kg body weight/day and 95th 5

percentile value of 4.86 mg/kg body weight/day), (FSANZ, 2004). In Brazil however, consumption of aspartame was negligible with more of cyclamate and saccharin been consumed due to cost advantages (Toledo and Ioshi, 1995).

2.3

THE CHEMISTRY OF ASPARTAME Aspartame is a white, odourless, intensely sweet powder (Table 2.1) which is soluble in

water and ethanol but insoluble in fats or oils. Aspartame is composed of phenylalanine (50%), aspartic acid (40%) and methanol (10%). Aspartame is produced from the coupling of amino acids L-phenylalanine methyl ester and L-aspartic acid to give a dipeptide methyl ester (Burdock, 2005). The manufacturing process can progress in one of two ways, if coupling is done using the enzymatic reactions only the α form is produced, if done chemically both the sweet α form and the non sweet β form are produced, thereby requiring separation process to get the α form (Magnuson et al., 2007). The stability of aspartame has been called to question, aspartame is very stable under dry conditions, but degrades during prolonged heat treatment in aqueous solutions, breakdown results in loss of sweetness as the breakdown products are not sweet (Magnuson et al., 2007). Five degradation products of a 1% aqueous solution of aspartame stored at 37◦ C for 2 months at pH 4.6 were reported by Furda et al., (1975), these were 3-carboxymethyl-6-benzyl-2, 5- diketopiperazine (DKP), L-aspartyl-phenylalanine, L-aspartic acid, L-phenylalanine, and L-phenylalanine methyl ester (Furda et al., 1975).

6

Table 2.1: General description of aspartame.

Appearance

White Crystalline powder

CAS No

22839-47-0

Chemical formula

C14H18N2O5

EINECS No.

245-261-3

Functionality in food

Sweetener; sugar substitute; flavor enhancer

INS No.

951

Molecular weight

294.31

NAS No.

1013

Odour

Odourless

CAS

=

Chemical

Abstracts

Service;

EINECS

=

European

Inventory

of

Existing

Chemical/Commercial Substances; INS = International Numbering System; NAS = National Academy of Sciences. (Burdock, 2005; Magnuson et al., 2007).

7

Studies by Rowe revealed that the rate of degradation of aspartame in aqueous solutions is dependent largely on pH and temperature (Rowe et al., 2003), aspartame’s stability is best at between pH 4 and 5, with a half-life of over 250 days at 25◦C (Rowe et al., 2003). The pathway and by-product of aspartame degradation is also dependent on the pH of the solution (Prodolliet and Bruelhart, 1993). At neutral and alkaline pH, aspartame cyclizes to form DKP or hydrolyzes to α-aspartyl phenylalanine and methanol, also at this pH interconversion can occur between the two degradation products but at no time do they revert back to form aspartame. At acidic pH (usually less than 4 or 5), aspartame undergoes rearrangement to β-aspartame, also cleavage of its peptide bond can occur giving phenylalanine methyl ester and aspartic acid. β-aspartame itself can also undergo cleavage to produce β-aspartyl phenylalanine (Magnuson et al., 2007). Lastly, phenylalanine may be formed from hydrolysis of aspartyl phenylalanine or phenylalanine methyl ester (Bell and Labuza, 1994). The stability of aspartame has also been reported to be dependent on water-activity which is defined as the ratio of the vapour pressure of water in a solution to the vapour pressure of pure water (Vaclavik and Christian, 2003). The effect of water-activity on aspartame breakdown under varied pH conditions was investigated, aspartame degradation increased with water-activity. Dry foods are those with a water-activity below 0.6 as no known microbes can grow at this level (Bell and Labuza, 1994). Aspartame’s ability to remain stable in commercially sterilized dairy beverages has also been questioned, Prodolliet and Bruelhart (1993) studied the amount of aspartame and its decomposition products in 24 commercial food products using high-performance liquid chromatography (HPLC), breakdown products of aspartame were present at the highest amounts in dairy products, including commercially prepared fruit cream, three milk chocolate, malt beverages, and fruit yogurt; however, most foods contained within 90% of the amount of aspartame claimed to be contained in them. Thus they concluded that although some degradation

8

occurred, a large fraction of aspartame; over 90% was still retained (Prodolliet and Bruelhart, 1993). 2.3.1 DIKETOPIPERAZINE Diketopiperazine (DKP), the cyclization product of aspartame, is one of many ubiquitous cyclic dipeptide derivatives found in nature and protein-rich foods (Magnuson et al., 2007). Naturally occurring DKP is found in a wide variety of foods ranging from cereal grains to bread, hydrolyzed vegetable protein, cheeses, processed meats such as ham and hotdogs, fish, fish sauce, dried shrimp, dried squid, and beverages including cocoa, coffee, beer, and milk (Ginz and Engelhardt, 2000). The amount of DKP in aspartame-containing foods depends on the type of food or beverage, the pH, and the time and temperature of storage (Homler, 1984). Under dry conditions however, the conversion of aspartame to DKP is slow, at a rate of 5% per 100 h at 105◦C (Homler, 1984). DKP levels in commercial foods purchased from stores, including soft drinks, juices, and dairy products, ranged from less than 0.3 to 14% of the aspartame concentration (Prodolliet and Bruelhart, 1993; Kotsonis and Hjelle, 1996). The rate of degradation is also affected by the composition of the food product, such as the presence of oil, which enhances the rate of aspartame degradation (Bell and Labuza, 1994). The stability of aspartame is reported to be greatly enhanced (up to 42%) in the presence of β-cyclodextrin (Garbow et al., 2001), another sweetener.

2.4

METABOLISM OF ASPARTAME Aspartame is composed of a phenylalanine molecule, an aspartate molecule and a methyl

group esterified to the carbonic acid group of the phenylalanine. The metabolism of aspartame and the products of its metabolic breakdown in animals, healthy individuals, and in subjects with phenylketonuria (PKU) have been comprehensively studied (Lajtha et al. 1994). Aspartame is metabolised into phenylalanine (50%), aspartic acid (40%) and methanol (10%). The first two are known as amino acid isolates. 9

2.4.1 ABSORPTION, DISTRIBUTION AND ELIMINATION OF ASPARTAME Aspartame is metabolized in the gastrointestinal tract by esterases and peptidases into aspartic acid, phenylalanine, and methanol in the upper part of the small intestine; methanol is released by hydrolysis of the methyl ester by pancreatic chymotrypsin, and is immediately absorbed in the small intestine (Magnuson et al., 2007). Aspartame may be completely hydrolyzed to these three components in the lumen of the gastrointestinal tract (GIT) and subsequently absorbed into the general circulation or may undergo hydrolysis to methanol and aspartyl phenylalanine dipeptide, in which case, the dipeptide is absorbed into the mucosa cells of the mucosa cells of the GIT and then subsequently broken down into the constituent amino acids (Stegink, 1987). Available data indicate that aspartame does not enter into circulation prior to hydrolysis. Metabolism of the three breakdown products of aspartame metabolism have been shown to be similar to the metabolism of these components if given individually (Stegink, 1987). Initial studies on aspartame focused on the effects of ingesting single bolus of aspartame on plasma concentrations of aspartate and phenylalanine levels and blood methanol concentrations in normal adults. These studies were done with doses of aspartame approximating current levels of dietary exposure (4 and 10 mg/kg body weight), doses representative of premarketing projections of the high level intake and the ADI (34 and 40 mg/kg body weight respectively), and ‘abuse’ doses of 100, 150 and 200 mg/kg body weight (Stegink and Filer, 1996). The plasma phenylalanine concentrations in healthy adults administered various doses of aspartame were then compared to values obtained: (1) in the fasting and postprandial state; (2) in individuals who are heterozygous for PKU; and (3) in subjects with various forms of hyperphenylalaninaemia other than PKU (Stegink and Filer, 1996). The data showed that the plasma phenylalanine concentrations after single bolus doses (ranging between 4 and 50 mg/kg body weight) and repeated doses (30 and 69 mg/kg body weight given as 3 and 8 divided doses respectively) of aspartame were generally within the normal postprandial range for this amino acid and well below those measured in 10

subjects homozygous for PKU after ingestion of aspartame. The aspartate component of aspartame is rapidly metabolised and thus the plasma aspartate concentrations are not significantly elevated following aspartame doses of 34 to 50 mg/kg body weight, whereas plasma phenylalanine concentrations may increase depending on dose (Stegink, 1987). Methanol is also rapidly metabolised and blood levels are usually not detectable unless large bolus doses of aspartame (>50 mg/kg body weight) are administered. Animal studies conducted by Opperman and co-workers in the 1970s demonstrated that aspartame is first hydrolysed to aspartyl phenylalanine and methanol by intestinal esterases, possibly chymotrypsin and the aspartyl phenylalanine is then further broken down into phenylalanine and aspartic acid (Oppermann et al., 1973; Oppermann and Ranney, 1979). Phenylalanine enters the plasma free amino acid pool from the portal blood after partial conversion to tyrosine by hepatic phenylalanine hydroxylase. Aspartate is metabolized within the enterocyte via transamination producing oxaloacetate, thereby reducing the concentration of aspartate entering the portal circulation and plasma free amino acid pool (Stengink and Filer, 1996). Methanol however is not subject to metabolism within the enterocyte and rapidly enters portal circulation, here it is oxidized in the liver to formaldehyde. Enzymes involved are species specific. In the rat, the metabolism of methanol to formaldehyde is mediated though a catalase-peroxidase system, whereas in primates and humans, an alcohol dehydrogenase is responsible (Magnuson et al., 2007). Formaldehyde is further oxidized to formic acid by formaldehyde dehydrogenase. Formaldehyde has a half-life of only 1- 2 min with an average of 1.5 minutes, so it does not accumulate (Liesivuori and Savolainen, 1991). Formaldehyde is a deadly neurotoxin (Lee et al., 1994). Rodents do not develop metabolic acidosis during methanol poisoning, owing to their high liver folate content, and in order to create similar results in human beings, folate deficient rodents have been used to accumulate formate in order to develop acidosis in methanol poisoning (Eells et al., 2000). Formic acid is ultimately converted to CO2 and water, via the formation of 10- formyl tetrahydrofolate (Barceloux et al., 2002). 11

The metabolism of aspartame has been studied in mice (Oppermann and Ranney, 1979), rats (Fernstrom, 1989; Hjelle et al., 1992), rabbits (Ranney et al., 1975), pigs (Burgert et al., 1991), dogs (Karim and Burns, 1996), monkeys (Oppermann et al., 1973), and humans (Karim and Burns, 1996) following administration orally or parenterally. Several of this studies concluded aspartame is digested in all species in the same way as any peptide (Ranney et al., 1975), however Reynolds et al. (1980) reported that the metabolism of phenylalanine is faster in monkeys than in humans, as the rise in blood phenylalanine levels following administration of an acute oral dose of aspartame was lower in infant monkeys than expected in humans. This has been attributed to a higher rate of catabolism of amino acids due to a higher protein requirement. Consumption of 50 mg aspartame /kg body weight results in ingestion of 5 mg methanol/kg body weight (10% of Aspartame by weight is methanol), which is believed to be far less than the amount of methanol formed during consumption of many foods including fruits and vegetables (Garriga and Metcalfe, 1988). In addition, Davoli et al., (1986) found that following consumption of 34 mg /kg of aspartame as a bolus dose (approximately equal to 20 cans of soda), methanol levels in serum remained within the normal postprandial range. Studies have shown that both methanol and formaldehyde could be toxic to the brain (Jeganathan and Namasivayam, 1998). The toxic effects of methanol in humans have also been reported to be secondary to the accumulation of its metabolite formate (Tephly, 1999), while severity of clinical findings in methanol intoxication correlated better with formate levels (Osterloh et al., 1986). Formate is metabolized twice as fast in rat as in monkey (McMartin et al., 1978). The metabolism of formate is mediated through a tetrahydrofolate-dependent pathway (Eells et al., 2000). Humans and non-human primates are uniquely sensitive to methanol poisoning because of their low liver folate content (Johlin et al., 1987). There are profound differences in the rate of formate oxidation in different species which determine their sensitivity to methanol (Mcmartin et al., 1978). In non-human primates and humans, alcohol dehydrogenase mediates this reaction (Makar et al., 1990). In rats and other non-primate species, this reaction is mediated by catalase. The 12

microsomal oxidizing system is believed to be responsible for free radical generation. In addition to this, inhibition of cytochrome oxidase by formate leads to the generation of superoxide, peroxyl and hydroxyl radicals. Tephly (1999) also reported that the catalase system is also one of the major pathways for methanol oxidation in rat hepatocytes. Free methanol is created from aspartame when it is heated to above 30°C, this would occur when an aspartame-containing product is improperly stored or when it is heated. A number of animal studies have demonstrated that the metabolic breakdown products of aspartame are absorbed and metabolised similarly, whether they are given alone or derived from aspartame. The extensive presystemic metabolism of aspartame results in little or no parent compound reaching the general circulation (Lajtha et al., 1994).

2.5

ASPARTAME: IS IT SAFE? The possibility of harm arising from the use of artificial sweeteners continues to generate a

fair amount of news coverage and even more confusion for consumers. The FDA (US Food and Drug Administration) has designated them as safe; although a number of interest groups beg to differ, believing that research on artificial sweeteners is flawed, biased and does not account for how long-term use of the additives may impact health. The first insecurity shook the artificial sweetener market in 1970, when cyclamate was banned by FDA in USA for its alleged causative role of cancer in experimental animals (FDA, 1970; FSANZ, 2004). It took close to 20 years to withdraw from the market this ‘sweet poison’. Cyclamate is however still used in a number of countries usually in combination with other artificial sweeteners. Increasing demands for low calorie artificial sweetener saw the discovery of aspartame with promising potentials for human use. Since then worldwide research has been carried out on its safety in human. In spite of being projected as a potentially toxic compound by several studies, the use of aspartame still continues. Safety issues raised in the past about aspartame have included: (1) the possibility of toxicity from methanol, one of the breakdown products of aspartame; (2) elevations in plasma 13

concentrations of phenylalanine and aspartic acid, which could result in increased transport of these amino acids into the brain, altering the brain's neurochemical composition (Coulombe and Sharma, 1986), (3) the possibility of neuroendocrine changes, particularly increased concentrations in the brain, synaptic ganglia and adrenal medulla of catecholamines derived from phenylalanine and its hydroxylation product, tyrosine; and (4) a postulated link with epilepsy and brain tumours (Olney et al., 1996). The Scientific Committee for Food (SCF) continues to evaluate and regulate aspartame use, this was first done in 1984, subsequently in 1988, and then at its 107th meeting in June 1997, the SCF also examined the issue of alleged connection between aspartame and increase in incidence of brain tumours in America (SCF, 1997). Aspartame safety has also been considered by other bodies including the Joint Expert Committee on Food Additives (JECFA, 1980) the US Food and Drug Administration (FDA, 1984), and the UK Committee on Toxicity (COT, 1996). Toxicity data on aspartame were used by these groups to establish an acceptable daily intake (ADI) of 40 mg/kg body weight/day in Europeans and 50 mg/kg body weight/day in Americans. An ADI of 7.5 mg/kg body weight/day was also established for a minor cyclic dipeptide derivative of aspartame, a diketopiperazine (DKP), which is formed in some aqueous solutions (JECFA, 1980; SCF, 1997). Anecdotal reports on the toxic effects of aspartame are numerous, and various issues continue to be raised today, more than 30 years after its approval by the FDA. Concerns relating to possible adverse effects have been raised due to the metabolic components, phenylalanine, aspartic acid, diketopiperazine (DKP) and methanol (Trocho et al., 1998) as well as aspartame itself. Studies investigating the toxicity of aspartame and the cyclization product of aspartame, DKP, continue to this day. Almost all segments of human life and existence have been studied against aspartame use, acute, subchronic, and chronic bioassays, neurotoxicity studies, immunotoxicity studies, reproductive, teratogenic, and multigenerational bioassays in animals, as well as carcinogenic bioassays. Bacterial studies and in vitro cell culture studies have also been utilized to assess the toxicity of aspartame (Magnuson et al., 2007).The results of these studies continue to be 14

rallying points for debate. Divergence of opinion exists, fuelled largely by the possible absence of trust, high degree bias in the analysis and reporting of these studies regardless of the outcome, a number of these studies and reviews are sponsored by either manufacturers e.g Ajinomoto company (Magnuson et al., 2007), Searle Laboratories (Searle Laboratories, 1974), or groups that may or may not benefit financially. Acute toxicity studies with aspartame (Table 2.2) have been conducted using oral and intraperitoneal exposure routes with mice, rats and rabbits. Available data declare that no deaths or adverse effects were observed with oral dosages as high as 10 g/kg body weight. Acute toxicity studies were also conducted with DKP in mice, rats and rabbits, also no deaths or adverse effects were reported with oral dosages as high as 5 g/kg body weight (Magnuson et al., 2007). The acute toxicity of β-aspartame was also evaluated in mice and rats with oral doses of 2500 and 5000 mg/kg body weight (Kotsonis and Hjelle, 1996), as no deaths or adverse effects were observed, the LD50 of β-aspartame in mice and rats is greater than 5000 mg/kg.

15

Table 2.2: Published results of acute toxicity tests for aspartame Test Species

Route

Route

Test dose (mg/kg body weight

Mouse1 Mouse1, male Sprague-Dawley (Ha/ICR) Rat1 Rabbit male New Zealand Rat male Charles River

Oral Oral Oral

LD 50 LD 50 LD 50

>10,000 >5000 > 5000

Oral Oral

LD 50 LD 50

>10,000 >5000

Rat1

IP

1

IP

>1562

LD 50

>5000

Strain and sex not defined; LD50 = the dose that produces 50% lethality in the test population;

IP=intraperitoneal. (Adapted from Magnuson et al., 2007).

16

2.6

ASPARTAME: REGULATION AND APPROVALS Aspartame has been approved for use in food in more than 90 countries worldwide (Health

Canada, 2005), and has been added to over 6000 products worldwide (AFSSA, 2002). In recent review of the safety of aspartame use, the European Union’s Scientific Committee on Food maintained the established acceptable daily intake (ADI) of aspartame by humans at 40 mg/kg of body weight (body weight), while the U.S. Food and Drug Administration (FDA) established an ADI of 50 mg/kg body weight for aspartame (Code for Federal Regulatons, 2005). The European Food Safety Authority (EFSA) also evaluated results of a study on the long-term carcinogenicity potential of aspartame and confirmed the previously established ADI for aspartame (EFSA, 2006). Aspartame is approved by the FDA for use under the Code for Federal Regulation (CFR) CFR:172.804 as a flavour enhancer and general-purpose sweetening agent for foods in which standards of identity do not preclude such use and any food containing the additive must bear on its label the following statement: “Phenylketonurics: contains phenylalanine.” In addition, when aspartame is used as a sugar substitute for table use, the label must bear instructions not to use it in cooking or baking. Aspartame is also approved for use as an inactive ingredient in drug formulations (FDA, 2006).

2.7

ASPARTAME AND THE BRAIN Aspartame has been reported to be involved in brain pathologies (Olney et al., 1996)

resulting in brain tumors, memory loss, seizures, headaches confusion, personality disorders, visual difficulty (Tollefson and Barnard, 1992) and dizziness are some of the negative effects that have been reported (Johns, 1988). These concerns arose following a study by Olney and Ho (1970) that demonstrated the presence of hypothalamic injury in infant Swiss albino mice 5 hours after they received a large oral doses (1 g/kg body weight) of aspartic acid, glutamate, or 3 g/kg body weight of cysteine (Olney and Ho, 1970), Schainker and Olney in another study this time administering subcutaneous injections of aspartic acid to day-old Swiss albino mice for 4 days at a 17

dose of 15 mmol/kg body weight resulted in high mortality rates, hypothalamic lesions and obesity in survivors (Schainker and Olney, 1974). Pizzi and colleagues also reported incidences of increased body weight, behavioural changes and impairment in reproductive function in adult mice (180 day old) who had received subcutaneous injections of aspartic acid on days 2 to 11 of life at 2.2 g/kg body weight which was later increased to 4.4 g/kg body weight (Pizzi et al., 1978). Weaknesses associated with central nervous system motor neurone disease or death are usually clearly apparent compared to dicarboxylic amino acid-induced neurodegeneration which is clinically silent (Magnuson et al., 2007), hence the apprehension regarding the effects of aspartame on the brain, these reports has led to intensive research into the possible neurologic biochemical, behavioural and/or morphological changes following exposure to aspartame. In vitro studies using hippocampal slice preparation evaluated the effects of aspartame and reported that exposure of these slices to 0.01, 0.1, 1, and 10 mM aspartame resulted in potentiation of the response of hippocampal CA1 pyramidal cells, but had no apparent effect on local inhibitory systems. Exposure of slices of the hippocampus to aspartame did not block the establishment of induction of long-term potentiation at any of the dose studied despite the potentiation of pyramidal cell response (Magnuson et al., 2007). In other studies, using mouse NB2a neuroblastoma cells grown in culture in which differentiation can be induced with the addition of dibutyryl cyclic adenine monophosphate (cAMP) and the removal of serum from culture media resulting in growth of neurite-like extensions from the cell body. Inhibition of the growth of these amygdalae was employed as a measure of potential neurotoxicity of four food additives: aspartame, L-glutamic acid, brilliant blue, and quinoline yellow. These additives when added to the cell growth media were reported to inhibit induction of neurite growth (Magnuson et al., 2007). Tsakiris et al. (2006) reported that in vitro incubation of human erythrocyte membranes in the presence of methanol, aspartic acid and phenylalanine resulted in a dose-dependent reduction of acetylcholinesterase activity. Reports from animal studies have shown the development of 18

hypothalamic neuronal necrosis following administration of aspartame orally to young mice at 1–2 g/kg body weight and also when given drinking water containing a mixture of metabolites from aspartame consumption glutamate and aspartic acid (Olney and Ho, 1970). In another study using infant (macaque) monkeys who received either aspartame (2 g/kg body weight) or MSG (1–4 g/kg body weight by gastric tube, hypothalamic morphology remained normal in all animals both at the microscopic and ultrastructural level (Magnuson et al., 2007). In a study by Finkelstein and his team (1988) assessing the ability of aspartame in inducing neuronal necrosis in young mice (8-day old Swiss-Webster) given 750 or 1000 mg/kg body weight showed that simultaneous administration of carbohydrate (1 g/kg body weight) diet or prior injection of insulin had a protective effect despite little effect on plasma amino acid concentrations (Finkelstein et al., 1988). Aspartame’s ability to potentiate the induction of chemically or electrically induced seizures in rats was assessed as a measure of neurotoxicity using male Sprague-Dawley rats fasted for 16 h, and then given aspartame by oral gavage, Aspartame lowered the ED50 (effective dose that induced convulsions or seizures in 50% of the animals) of metrazol-induced convulsions from 65.9 mg/kg body weight to 50.7 mg/kg body weight when administered in a bolus dose of 1 g/kg body weight to fasted rats while no effect was observed when the same dose of aspartame was divided into 3 doses and delivered to fasted rats equally spaced over 120 minutes or when the dose was delivered to non-fasted rats (Magnuson et al., 2007) Similar results were found with administration of equimolar amounts of phenylalanine, but aspartic acid, methanol and leucine were inactive. Diomede et al. (1991) also compared the ability of aspartame to enhance metrazolinduced seizures in two strains of mice (CD1 and DBA/2J), COBS guinea pigs and SpragueDawley rats, he discovered that at doses up to 2000 mg/kg body weight, no potentiation of seizures was observed in either mice strain or guinea pigs, rats given 1000 mg/kg body weight however had a higher incidence of metrazol-induced seizures compared to vehicle. The authors were of the opinion that species differences in the rate of conversion of phenylalanine to tyrosine 19

may have been responsible as at the high doses used, saturation of hepatic phenylalanine hydroxylase may occur (Diomede et al., 1991). Olney et al. (1996) published an article on a possible link between the increase in the frequency of brain tumours in humans and the consumption of aspartame in the United States, based on the data from the National Cancer Institute (10% of the population) from 1975-1992, the authors concluded that there was a significant increase in the frequency of brain tumours in the mid-1980s, that is to say the period following when aspartame came into the market. The conclusions of this epidemiological study have been criticised by a number of scientists who questioned the methodology, the use of the data and their interpretation (Levy and Hedekker, 1996; Linet et al., 1999). One of the major criticism is that the authors only took into account the frequency of brain tumours during a selected period (1975- 1992). When all the epidemiological data were used (1973-1992) the critics reached a different conclusion, the frequency of brain cancers increased in 1973 and stabilised from the mid-1980s (Levy and Hedekker, 1996). Furthermore, they believed that Olney and his colleagues did not provide any quantitative or qualitative relationship between the exposure of the population to aspartame and the observed frequency of brain tumours, finally, an increase in the incidence of the tumours reported by Olney could have many causes including, among others, improvements in diagnostic methods (Modan et al., 1992). Gurney et al., (1997) also published the results of a case-control study on the relationship between the consumption of aspartame and the frequency of brain tumours. The study covered 56 patients affected by tumours in childhood and 94 controls. According to these authors, “no relationship could be established between the consumption of aspartame and the frequency of brain tumours.” Short-term studies have been conducted to assess the effect of aspartame on learning or memory, regardless of subject (humans or animals), Moser and colleagues suggested a relationship between aspartame consumption and memory loss (Moser, 1994), although quite a number of other researchers have concluded the absence of any such relationships and have reported no adverse effects of aspartame on memory (Magnuson et al., 2007). Holder (1989) showed that 50 days of NutraSweet had no 20

effect on reflex or spatial memory development. In another study by Leon et al. (1989) no persistent changes in vital signs, body weight or standard laboratory tests were seen in subjects receiving aspartame for 24 weeks (Leon et al., 1989); however, extensive memory testing was not done. A few chronic studies have implicated aspartame consumption in learning or and memory, some of such studied showed that administration of aspartame as 9% of the diet for 13 weeks altered learning behaviour in male rats. Using a much lower daily dose of aspartame, in another study this time using pregnant guinea pigs that received aspartame throughout gestation and demonstrated the aspartame-treated pups showed a disruption of odour-associative learning. The issue is of great interest, from the point of view of human health, considering that pregnant women consume a highly varied array of products that contain aspartame, monosodium glutamate, and hydrolyzed protein too, used as food additives (Magnuson et al., 2007).

2.7.1 ASPARTAME AND BRAIN LEVELS OF AMINO ACIDS Concerns have been voiced regarding possible effects of aspartame consumption on brain amino acid levels and brain function. Headaches, insomnia and seizures encountered following consumption of aspartame are believed to be secondary to changes in regional brain concentrations of catecholamines, which include norepinephrine, epinephrine and dopamine (Coulombe and Sharma, 1986). Administration of aspartame can elevate brain levels of phenylalanine and, especially tyrosine in the rat (Wurtman, 1983; Yokogoshi, 1984). Changes in the plasma levels of amino acids individually in response to aspartame may be of limited value in assessing the potential effect of aspartame on brain uptake of the amino acids, because cells of the blood–brain barrier contain high levels of 20 or more specific transport systems that regulate the flux of key solutes from blood into brain interstitial fluid and cerebrospinal fluid and back out again (Magnuson et al., 2007).

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There are four types of transporters of amino acids. These are: (1) System L, which mediates high-affinity, sodium-independent uptake of zwitterionic amino acids with “large, neutral” side chains, including L-leucine, L-phenylalanine, L-tryptophan, L-tyrosine, L-isoleucine, L-methionine, and L-valine; (2) System y+, which mediates moderate-affinity, sodiumindependent uptake of amino acids with cationic side chains, including L-arginine, L-lysine, and L-ornithine; (3) System T, which mediates high-affinity, low capacity transport of thyroid hormones (T3 and T4); and (4) System x−, which mediates sodium-independent, high-affinity uptake of amino acids with anionic side chains, including L-glutamate and L-aspartic acid. Due to transport saturation, individual amino acids must compete for transport. Transport saturation makes the brain amino acid delivery selectively vulnerable to large imbalances in plasma amino acid concentration such as those that occur in the hyperaminoacidemias, e.g., phenylketonuria and maple syrup disease. Thus, the brain uptake of any one amino acid increases only when the ratio of this amino acid to the other amino acids is increased (Magnuson et al., 2007). Some earlier studies have reported that aspartame or its metabolites are restricted from getting to the brain because of the blood brain barrier (BBB), a compromised BBB (altered lipid-mediated transport or active carrier transport) however will result in the transport of excitotoxins (aspartate) across BBB and within the cerebrospinal fluid causing several adverse reactions to occur (Humphries et al., 2008). The nerves will be stimulated to fire excessively by the excitotoxins, the offset of induced, repeated firing of the neurons mentioned above will require normal enzymes, which are negated by the phenylalanine and aspartic acid present in aspartame and the neurons become compromised from diminishing intracellular ATP stores; the presence of formaldehyde, and intracellular calcium uptake (e.g. phenylalanine binds to NMDA receptor, not glutamate, thus altering calcium channels); cellular mitochondrial damage; destruction of the cellular wall; and subsequent release of free radicals (Humphries et al., 2008). These preceding reactions potentiate oxidative stress and neurodegeneration. Secondary damage is caused by the toxic by-products, which in turn increases capillary permeability, continuing to destroy the surrounding nerve and 22

glial cells, thus further obstructing enzyme reactions and promoting DNA structural defects, cellular death occurs over the next 1–12 h. Aspartate affects all parts of the developing brain, the hypothalamic regions that lack blood brain barrier, extrahypothalamic regions (hippocampus, choroids plexus and cortex), because the developing brain is extremely vulnerable to the excitotoxic insults. There is some evidence that it may actually be concentrated within the brain with prolonged exposures (Humphries et al., 2008). There are also several conditions under which the blood-brain barrier (BBB) is made incompetent. Before birth, the BBB is incompetent and will allow glutamate, aspartate and phenylalanine to enter the brain and for a considerable period after birth the barrier remains incompletely developed, also with increasing age the barrier system becomes more porous, allowing excitotoxins in the blood to enter the brain. So there are numerous instances under which excitotoxin food additives can enter and damage the brain (Humphries et al., 2008). Recent experiments have shown that aspartate can open the barrier itself. Another system used to protect the brain against environmental excitotoxins, is a system within the brain that binds the glutamate molecule (called the glutamate transporter) and transports it to a special storage cell (the astrocyte) within a fraction of a second after it is used as a neurotransmitter. This system can be overwhelmed by high intakes of monosodium glutamate, aspartame (Humphries et al., 2008) and other food excitotoxins.

2.7.1.1 Aspartame and Aspartic Acid Studies have shown that aspartate (component of aspartame molecule) is a powerful amino acid neurotransmitters that clearly play a pivotal role in neuronal differentiation, migration and survival in the developing brain, neuronal plasticity, in the formation of synapses and neuronal circuitry, long-term potentiation and depression, and both normal learning and addictive behaviour (Olney et al., 1996). Aspartate in conjunction with glutamate act postsynaptically on three families of ionotropic receptors, named after their preferred agonists, N-methyl-D-aspartate (NMDA), α23

amino-3-hydroxy-5-methyl-4-isoxazole propionicacid (AMPA) and kainate (KA). These receptors all incorporate ion channels that are permeable to cations, although the relative permeability to Na+ and Ca2+ varies according to the family and the subunit composition of the receptor. Some studies have suggested that NMDA may have a trophic influence on developing neurons (Olney et al., 1996). NMDA has been reported to enhance neuritis outgrowth and cell survival in tissue culture, NMDA receptor activation might also mediate programmed cell loss (apoptosis) that is present during normal development, (Lynch, 1994). In the last decades L-aspartate and/or glutamate have become recognized as molecules in the CNS that can have both a protective effect as well as a neurotoxic potential (Lynch, 1994). Aspartate in the brain in normal, physiological conditions, serves vitally important metabolic mechanisms (in protein and nucleic acids biosynthesis, in tricarboxylic acids chain), and may also play an important role in developmental plasticity. NMDA receptors appear to be crucial for determining the relative positions of synaptic inputs during early development and may be a prerequisite for the formation of ocular-dominance columns in the visual cortex (Meldrum, 2000). In some other instances, when these amino acids exist in concentrations above normal levels, they harbour treacherous neurotoxic potential. Excess aspartate during embryogenesis has been shown to reduce dendrite length and suppress axonal outgrowth in hippocampus neurons. It is interesting to note that aspartate can produce classic toxicity in the immature brain even before the glutamate receptors develop. High excitotoxins levels can also affect astroglial proliferation as well as neuronal differentiation. It appears to act via the phosphoinositide protein kinase C pathway, (Meldrum, 2000). These aspects cause very important consequences on the following prepubertal, juvenile stages of development of animals. The studies by Olney (1969), Fernstrom, (1989) revealed that after treatment with aspartate or glutamate in the gestational period, the infant rodents are born with incomplete myelinization, aspartate and glutamate being recognized excitotoxins literally excite the neurons to death. More recent molecular studies have disclosed the mechanism of this destruction in some detail (Ho et 24

al., 2003). Subsequent studies have shown that aspartate, and other excitatory amino acids, attach to a specialized family of receptors (NMDA, kainate, AMPA and metabotrophic) which in turn, either directly or indirectly, opens the calcium channel on the neuron cell membrane, allowing calcium to flood into the cell. If unchecked, this calcium will trigger a cascade of reactions, including free radical generation, eicosanoid production, and lipid peroxidation, which will destroy the cell. With this calcium triggered stimulation, the neuron becomes very excited, firing its impulses repetitively until the point of cell death, hence the name excitotoxin (Ho et al., 2003). The activation of the calcium channel via the NMDA type receptors also involves other membrane receptors such as the zinc, magnesium, phencyclidine and glycine receptors. In many disorders connected to excitotoxicity, the source of the glutamate and aspartate is indigenous. It is known that when brain cells are injured, they release large amounts of glutamate from surrounding astrocytes, and this glutamate can further damage surrounding normal neuronal cells. This appears to be the case in stroke, seizures and brain trauma. But, food borne excitotoxins can add significantly to this accumulation of toxins.

2.7.1.2 Aspartame and Phenylalanine Phenylalanine plays an important role in amino acid metabolism and protein structuring in all tissues, it is also a precursor for tyrosine (Hawkins et al., 1988), DOPA (dihydroxy phenylalanine), dopamine, norepinephrine and epinephrine (Ganong, 1997). Phenylalanine is also important in neurotransmitter regulation (Caballero and Wurtman, 1988). Phenylalanine metabolism progresses in one of two ways; it can be converted into tyrosine in the liver by the enzyme phenylalanine hydroxylase, or in the case of increased blood levels of phenylalanine bind to a large neutral amino acid transporter (NAAT) to be carried across the blood–brain barrier (BBB) directly into the brain (Caballero and Wurtman, 1988). High daily consumption of aspartame would result in an increase in blood levels of phenylalanine because it makes up 50% of it breakdown product. Phenylalanine in turn gets converted to tyrosine or cross the BBB as is. 25

Tyrosine itself gets carried across the blood brain barrier by binding to the NAAT transporter and here it is converted into dihydroxyphenylalanine (DOPA) by the action of tyrosine hydroxylase and this itself is an inhibitory neurotransmitter (Humphries et al., 2008). Following the administration of aspartame to humans, increases in blood levels of both phenylalanine and tyrosine have been reported (Filer and Stegink, 1988; Fernstorm, 1989), further studies have shown that increases in both phenylalanine and tyrosine potentially disrupt a wide range of processes in the brain (Humphries et al., 2008), including amino acid metabolism, protein structure and metabolism, nucleic acid integrity, neuronal function and endocrine balances. Studies by Mehl-Madrona (2005) showed that aspartame changes the dopamine level in the brain especially in people affected by Parkinson disease. In natural occurring foods that contain large amounts of amino acids, e.g. meat which contain a chain of 80–300 amino acids, of which 4% are phenylalanine. This chain also includes the amino acid valine. Valine inhibits the transport of phenylalanine into the brain across the BBB. In aspartame, phenylalanine makes up 50% of the molecule; thus, in a can of diet soda, which contains 200 mg aspartame, 100 mg is phenylalanine. No valine is present in aspartame to block the entry of toxic levels of phenylalanine into the brain, thus resulting in lowered concentrations of dopamine and serotonin owing to neutral amino acid transporter (NAAT) occupation by phenylalanine (Humphries et al.2008). Comparing the results of human studies to those of other species, physiologically, the animals tested for phenylalanine toxicity are approximately 60 times less sensitive than human beings. The differences in enzyme concentrations of the species suggest that animals studied are more sensitive to the more common ethanol found in alcoholic beverages.

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2.8

NEUROBEHAVIOUR: ANIMAL MODELS OF BEHAVIOUR McKinney, (1984) defined animal models as experimental preparations developed in a

specie solely for the purpose of studying phenomena occurring in another species. Kaplan (1973) also suggested that a model may be valid if it has the same structure as the human behaviour or pathology being studied; it is believed that whenever a relation exists between two elements of the animal model, a corresponding association may exist amongst corresponding elements of the human behaviour. For an animal model to be suitable for research it has to have predictive validity (pharmacological correlation), face validity (isomorphism) and construct validity (homology and similarity of underlying neurobiological mechanisms) (McKinney and Burney, 1969; Triet, 1985). Predictive validity implies that the animal model should be sensitive to clinically effective pharmacological agents. Anxiolytic agents should show reduced anxiety, conversely, anxiogenic compounds should elicit opposite effects, while agents that have no effect in the clinic should have no effect in these tests. It is important to note that this involves that a given model may include both variables that are increased by anxiety as well as variables that are decreased by anxiety. For example, when an animal is confronted with a potent source of danger, it displays increased risk assessment behaviours and decreased exploratory activity (Belzung and Griebel, 2001). In many cases, only the second category of variables are recorded so that an increase in anxiety can be confounded with a non-specific inhibition of activity, such as sedation, ataxia, myorelaxation, preictal prostration or even toxic effects induced by the treatment. Face validity implies that the behavioural response observed in the animal model should be identical to the behavioural and physiological responses observed in humans. This indicates that the expression of a given emotion is supposed to be similar across species. Construct validity relates to the similarity between the theoretical rationale underlying the animal model and the human behaviour. This requires that the etiology of the anxiety behaviour for example and the biological factors underlying anxiety may be similar in animals and humans (Belzung and Griebel, 2001). 27

2.8.1 RODENT EXPLORATORY BEHAVIOUR Exploratory behaviour can be defined as the tendency to explore or investigate a novel environment. It is considered a motivation not clearly distinguishable from curiosity; other terminologies used to describe this behaviour include novelty-seeking behaviour or noveltyinduced behaviours. Exploratory behaviour is a prerequisite of the putative spatial representation system in the mammalian brain (O’Keefe and Nadal, 1978), it refers to a broad category of behaviours that function to provide an organism with information about its external environment, representing a fundamental characteristic of all living species (Berlyne, 1960). Behavioural studies reveal that in the course of exploration of the environment, various species are predisposed to return to well known locations termed home base resulting in looping or round-trip behaviour. Exploratory behaviour was thought in the earlier part of the 20th century to be motivated only by the need for survival which is dictated solely by food-seeking behaviour, need for procreation and the avoidance of injury (Cannon, 1953). Research however suggests that exploration persists even in the presence of stimuli that are neither biologically beneficial nor harmful (Welker, 1961). Behavioural theorists have proposed that animals engage in exploration up to a level of stimulation that will optimize the function of the brain in the absence of any explicit threat or danger (Berlyne, 1960). Berlyne proposed that exploratory behaviour can be divided into two distinct categories: (1) inspective exploration, when an organism will examine a particular location or source in response to a specific need for information; (2) diversive exploration, a general drive to seek out stimulation anywhere in the environment, resulting in contact with distant stimuli. More recent works have emphasized the theory that novel environments create conflict by inducing both approach and avoidance behaviours, pitting the desire to explore or approach, against the tendency to fear and thus avoid novelty (Dulawa et al., 1999). The assessment of locomotor and exploratory behaviour in rodents is one of the most widely used behavioural methods to determine the effects of genetic, anatomical, physiological, and pharmacological manipulations. Motor activity in rodents has been measured using a number 28

of behavioural paradigms but the oldest and most common paradigm is the Open field test (Robbins, 1979). The Open field apparatus (Plate 2.1a and 2.1b) is usually a rectangular novel open space either enclosed by a surrounding wall or elevated above the floor to prevent escape. The Open field test has been validated for use in a number of mammalian species from small to large. The animal usually a small mouse, gerbil or rat is placed in the field for a fixed time interval and a number of activity variables are quantified, these parameters include the distance covered per unit time, the number of regions visited (central or peripheral), rearing, latency to initial movement, stereotypic behaviours such as sniffing or grooming, and physical responses such as defecation or urination. The reliability of the test is critically dependent on uniform experimental conditions (Henry et al., 2010) including how rodents are handled and housed prior to the test, the degree of illumination, the extent of ambient noise, the time of testing during the light-dark cycle, and the visibility of the experimenter in the testing room (Gould et al., 2009). An observer (either present in the test room or remotely viewing a video) can manually score rodent transitions between regions and quantify peripheral versus centre entries (Gould et al., 2009). To avoid problems associated with direct observation of rodents and simplify data collection, a number of automated Open field instruments have been developed, including photobeam and video recording systems (Geyer et al., 1986; Sanberg et al., 1985; Vorhees et al., 1992). Rodent behaviour in the open field test can be interpreted in a number of ways and has been described by different groups as representing diversive and inspective exploration, arousal, inhibition, and anxiety (Archer, 1973; Robbins, 1979; Geyer et al., 1986).

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Plate 2.1a: Mouse Open Field Box (adapted from www.ugobasile.com)

Plate 2.1b: Mouse Open Field Box

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Recent studies revealing degree of organization involved in exploratory behaviour have helped to broaden our knowledge of novelty-seeking behaviours in rodents (Drai and Golani 2001; Drai et al., 2001). When placed in a large open field, rodents adopt certain locations as home bases in which they linger, turn, rear and groom. In addition, they make periodic outward trips from a home base that are slow, circuitous, and marked by a number of stops and rapid return trips back to the home base. Locomotor behaviour also consists of movements at a number of speeds. Exploratory behaviour in rats, which may initially appear unstructured, is organized and divisible into components that are organized in relation to a reference point such as a real or virtual home base (Eilam and Golani 1989). The exploratory behaviour of mice is also organized, but establishment of a home base is more obvious in mice than in rats (Drai and Golani 2001).

2.8.2 ANXIETY BEHAVIOUR Fear and/ or anxiety can be defined as the response of a subject to real or imagined threats that may impair its homeostasis. This responses may be physiological (increase in heart rate, blood pressure), or behavioural (inhibition of ongoing behaviours, scanning, avoidance of the source of danger). When this response is excessive or maladaptive, it involves ‘pathological’ anxiety. Over the past three decades, a number of tests have been developed with the possibility of face, construct and/or predictive validity as animal models of anxiety disorders (Weiss et al., 2000). Most of these models involve exposure of subjects to external (e.g. cues earlier paired with foot shock, bright light, predator) or internal (e.g. drug states) stimuli that are assumed to be capable of inducing anxiety in animals (Belzung and Griebel, 2001). The Elevated plus maze (EPM) (Plate 2.2) is used to assess anxiety-related behaviours in rodents. It is used as a screening test for putative anxiolytic or anxiogenic compounds and as a tool in neurobiological anxiety research. The EPM apparatus is a plus-shaped apparatus with two open and two enclosed arms, each with an open roof, elevated from the floor by 40–70 cm. The model is premised on rodents' aversion of open spaces (Pellow et al., 1985; Carobrez and Bertoglio, 31

2005). Anxiolysis or anxiogenesis is measured in the plus maze by the proportion of time spent in the open arms (time in open arms/total time in open or closed arms), and or the proportion of entries into the open arms (entries into open arms/total entries into open or closed arms). Total number of arm entries and number of closed-arm entries are employed as measures of general activity (Hogg, 1996).

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Plate 2.2: Mouse Elevated plus maze (Chandi Touma®, Max Planck institute)

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2.8.3

SPATIAL LEARNING AND MEMORY Eric Kandel (2000) described learning as a process by which knowledge about the world

around us is acquired. Learning refers to a more or less permanent change in behaviour which occurs as a result of practice, memory however is defined as the process by which that knowledge of the world is encoded, stored and later retrieved (Kandel, 2000). Memory is a phase of learning. Learning in most mammals can be described in three stages: (1) acquisition, wherein a new activity is acquired or new verbal material memorized or memorizes verbal material. (2) retention which involves pondering on the new acquisition for a period of time; and (3) recall which enables one to reproduce the learned act or memorized material. Spatial memory is the part of memory responsible for recording information about one's environment and its spatial orientation, it is required to navigate around a familiar places. A rat's spatial memory is needed to learn the location of food at the end of a maze. It is often argued that in both humans and animals, spatial memories are summarized as a cognitive map. Spatial memory has representations within working, short-term and long-term memory. Spatial learning and memory are important for navigation and formation of episodic memories (Kandel, 2000). Short-term spatial memory (STM) can be described as a system allowing one to temporarily store and manage information that is necessary to complete complex cognitive tasks. Tasks which employ short-term memory include; 1) learning 2) reasoning, and 3) comprehension (Johnson and Adamo, 2010). Spatial memory is a cognitive process that enables identification of different locations as well as spatial relations between objects. Working memory (WM) is a limited capacity system which allows one to store and process information temporarily (Ang and Lee, 2008). This temporary store enables one to complete or work on complex tasks while being able to keep information in mind. The most recent evaluation of the Baddeley and Hitch multi-component model of working memory (Jones et al., 1995), suggests that there are four subcomponents to WM, namely the phonological loop; the visuo-spatial sketch pad; the central executive; and the episodic buffer (Ang and Lee, 2008). Spatial memory recall is built on a hierarchical structure 34

hinged on the fact that people remember the general layout of a particular space and then "cue target locations" located within that spatial set (Chun and Jiang, 1998), this includes an ordinal scale of the different features needed in the formation of a cognitive map (McNamara et al., 1989). A cognitive map is "a mental model of objects’ spatial configuration that permits navigation along optimal path between arbitrary pairs of points." This mental map is premised upon two fundamental bedrocks: layout, also known as route knowledge, and landmark orientation. Layout is potentially the first method of navigation that people learn to utilize; its workings reflect our most basic understandings of the world (Newman et al., 2007). Some animals also need spatial memory to navigate their surrounding, it has been shown that certain species of paridae and corvidae (such as the black-capped chickadee and the scrub jay) are able to use spatial memory to remember where, when and what type of food they have stored (Bird et al., 2003). The Y maze, radial arm maze and Morris water maze (Morris 1981) are models for studying memory. Mazes are typically designed with a centre platform and a varying number of arms (Cole and ChappellStephenson, 2003). Y Maze (Plate 2.3) spontaneous alternation is a behavioural test that measures the willingness of rodents to explore new environments. Rodents typically prefer to investigate a new arm of the maze rather than return to one previously visited. The hippocampus and medial entorhinal cortex (MEC) are key brain areas for spatial learning and memory. The hippocampus provides animals with a spatial map of their environment (O'Keefe and Dostrovsky, 1971). It stores information regarding non-egocentric space which allows for viewpoint manipulation from memory. It is however, important for long-term spatial memory of allocentric spaces (Ramos, 2000). The hippocampus makes use of reference and working memory and has the important role of processing information about spatial locations.

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Plate 2.3: Mouse Y maze (©2015 Stoelting Co.)

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2.9

THE BRAIN The brain, (Plate 2.4) a bilaterally symmetric, soft, gelatinous structure surrounded by its

meninges and enclosed in its bony cranium, is continuous with the spinal cord at the foramen magnum at the base of the skull. At birth, the human brain weighs less than 400 g, but by the beginning of the second year of life it has more than doubled in weight to 900 g. The adult brain weighs between 1,250 and 1,450 g and demonstrates a gender differential, since brains of males generally weigh more than those of females however in adults, the ratio of brain to body weight is greater in females than in males and that the increase in weight is due more to the proliferation of neuroglia than to the mitotic activity of neurons. The adult mouse brain weighs between 0.35 to 0.4 g with a body weight to brain ratio of 40:1 (Nieuwenhuys et al., 1998). Compared to the adult human brain which has apparent lobular organization and prominent gyri and sulci the adult mouse brain is not prominently lobed and is lisencephalic (absence of sulci and gyri), the neural tissue appears pale coloured due to the high lipid content of the myelin of the white matter. The amount of white matter increases as a cubic function of the animal’s size while the gray matter increases as the square (Hagan et al., 2012) During embryogenesis the brain is subdivided into five continuous regions, from rostral to caudal: the telencephalon, diencephalon, mesencephalon, metencephalon, and myelencephalon, these regions fold on one another as the brain grows in size and complexity. In the adult brain, only three regions are clearly visible, and these are the cerebrum, cerebellum, and part of the brainstem.

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Plate 2.4: Human and mouse brain (Cryan and Holmes, 2005)

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2.9.1 THE CEREBRUM The cerebral hemispheres are narrower posteriorly at the occipital pole than anteriorly at the frontal pole. They are large, oval structures that superficially resemble the surface of a shelled walnut. The midline longitudinal cerebral fissure, occupied in life by the falx cerebri, incompletely separates the two cerebral hemispheres from one another. The floor of the cerebral fissure is formed by the corpus callosum, a large myelinated fiber tract that forms an anatomical and functional connection between the right and left hemispheres (Patestas and Gartner, 2013). The surface of the cerebral hemisphere is composed of a highly folded collection of gray matter, known as the cerebral cortex. This folding increases the surface area and presents elevations, gyri, depressions, sulci. Deep to the cortex is a central core of white matter that forms the bulk of the cerebrum and represents fibre tracts, supported by neuroglia, ferrying information destined for the cortex and cortical responses to other regions of the central nervous system (CNS) (Patestas and Gartner, 2013). Buried within the mass of white matter are collections of neuron cell bodies, some of which are lumped together under the rubric of basal ganglia, even though, technically, they are nuclei. Large collections of gray matter are also present in the diencephalon, namely, the epithalamus, thalamus, hypothalamus, and subthalamus. The cerebrum is a hollow structure and the cavities within the cerebral hemispheres are called the right and left lateral ventricles, which communicate with the third ventricle via the interventricular foramen (foramen of Monro). The two lateral ventricles are separated from one another by two closely adjoined non-nervous membranes, each known as a septum pellucidum. Ependymal cells line each lateral ventricle, and protruding into each ventricle is a choroid plexus that functions in the production of cerebrospinal fluid (Patestas and Gartner, 2013). Each cerebral hemisphere is subdivided into five lobes: the frontal, parietal, temporal, and occipital lobes, and the insula. Additionally, the cortical constituents of the limbic system are also considered to be a region of the cerebral hemisphere and some consider it to be the sixth lobe, the limbic lobe. Viewed from the side, each cerebral hemisphere resembles the shape of a boxing 39

glove, where the thumb is the temporal lobe and is separated from the parietal lobe by the lateral fissure (fissure of Sylvius). The floor of the lateral fissure is formed by the insula (island of Reil) that is hidden by the frontal, parietal, and temporal opercula, regions of the same named lobes. Although the geographic distributions of many of the sulci and gyri are relatively inconsistent, some regularly occupy specific locations, are recognizable in most brains, and are named. The sulci are generally smaller and shallower than the fissures, and one of these, the central sulcus (central sulcus of Rolando), separates the frontal lobe from the parietal lobe. The division between the parietal and occipital lobes is not readily evident when viewed from the lateral aspect because it is defined as the imaginary line between the preoccipital notch and the parieto-occipital notch. However, it is clearly delimited on the medial aspect of the cerebral hemisphere, where the boundary between these two structures is the parieto-occipital sulcus and its continuation, the calcarine fissure (Patestas and Gartner, 2013). The basal ganglia, called ganglia even though they are nuclei, are large collections of cell bodies that are embedded deep in the white matter of the brain. These soma include those deep nuclei of the brain and brainstem which, when damaged, produce movement disorders. Thus the basal ganglia are composed of the caudate nucleus, lenticular nucleus (putamen and globus pallidus), subthalamic nucleus of the ventral thalamus, and the substantia nigra of the mesencephalon (the caudate nucleus and the putamen together are referred to as the striatum). These nuclei have numerous connections with various regions of the CNS; some receive input and are categorized as input nuclei, some project to other regions and are referred to as output nuclei, whereas some receive input, project to other regions of the CNS, and have local interconnections and these are known as intrinsic nuclei (Patestas and Gartner, 2013). The diencephalon, interposed between the cerebrum and the midbrain, has four regions: the epithalamus, thalamus, hypothalamus, and subthalamus. The right and left halves of the diencephalon are separated from one another by a narrow slit-like space, the ependymal-lined third ventricle. Rostrally, the interventricular foramina (of Monro) leads from the lateral ventricles 40

into the third ventricle, whereas caudally, the third ventricle is connected to the fourth ventricle by the cerebral aqueduct (of Sylvius) (Patestas and Gartner, 2013). The epithalamus, composed of the pineal body, stria medullaris, and habenular trigone, constitutes the dorsal surface of the diencephalon. The right and left thalami compose the bulk of the diencephalon and form the superior aspect of the lateral walls of the third ventricle. The two thalami, structures composed of numerous nuclei, are connected to each other by a bridge of gray matter, the interthalamic adhesion (massa intermedia). Some of the nuclei of the thalamus form distinctive bulges on its surface, namely the pulvinar and the medial and lateral geniculate bodies. The boundary between the thalamus and the hypothalamus is marked by a groove, the hypothalamic sulcus, located along the lateral walls of the third ventricle. Structures associated with the hypothalamus are the pituitary gland and its infundibulum, the tuber cinereum, and the two mammillary bodies. The subthalamic nuclei and fibre tract form the subthalamus (Patestas and Gartner, 2013).

2.9.1.1 Histology of the Cerebral Cortex The cerebral cortex is well endowed with neurons, neuroglia, nerve fibres, and a rich vascular supply. The arrangement of the three types of neurons that populate the cortex, pyramidal cells, stellate neurons, and fusiform neurons permit the classification of the cortex into three types: the archicortex (allocortex), mesocortex (juxtallocortex), and neocortex (isocortex) (Patestas and Gartner, 2013). The archicortex, phylogenetically the oldest region, is composed of only three layers and is located in the limbic system. The mesocortex, phylogenetically younger, is composed of three to six layers, and is located predominantly in the insula and cingulate gyrus. The neocortex, phylogenetically the youngest region of the cerebral cortex, is composed of six layers and comprises the bulk of the cerebral cortex. Although the cerebral cortex is arranged in layers, superimposed upon this cytoarchitecture is a functional organization of cell columns. Each cell column is less than 0.1 mm in diameter, is perpendicular to the superficial surface of the cortex, passes through each of the six cortical layers, and is composed of neurons with similar functions. 41

All neurons of a single column respond to like stimuli from the same region of the body (Patestas and Gartner, 2013). The organization of the six layers of the neocortex is known as its cytoarchitecture, where each layer has a name and an associated Roman numeral. The central core of white matter that forms the substance of the cerebrum is composed of myelinated nerve fibres of varied sizes and their supporting neuroglia. These fibres may be classified into the following three categories: commissural, projection, and association fibres. Commissural fibres (transverse fibres) interconnect the right and left cerebral hemispheres. There are four bundles of commissural fibres, the corpus callosum, anterior commissure, posterior commissure, and hippocampal commissure. The largest group of the commissural fibres, the corpus callosum, is comprised of four regions: the anteriormost rostrum, the curved genu, the relatively flattened body, and its posteriormost region, the splenium. The corpus callosum connects the neocortex of the right hemisphere with that of the left. The anterior commissure connects the right and left amygdalas, the olfactory bulbs, and several cortical regions of the two temporal lobes. The posterior commissure connects the right and left pretectal region and related cell groups of the mesencephalon. The hippocampal commissure (commissure of the fornix) joins the right and left hippocampi to one another (Patestas and Gartner, 2013). Projection fibres are restricted to a single hemisphere and connect the cerebral cortex with lower levels, namely the corpus striatum, diencephalon, brainstem, and spinal cord. The majority of these fibres are axons of pyramidal cells and fusiform neurons. These fibres are component parts of the internal capsule, which is subdivided into the anterior limb, genu, posterior limb, retrolentiform, and sublentiform regions. The projection fibres may be subdivided into corticopetal and corticofugal fibers. Corticopetal fibres are afferent fibres that bring information from the thalamus to the cerebral cortex. They consist of thalamocortical fibres. Corticofugal fibres are efferent fibres that transmit information from the cerebral cortex to lower centres of the brain and

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spinal cord. They consist of the corticobulbar, corticopontine, corticospinal, and corticothalamic fibres (Patestas and Gartner, 2013). Association fibres, also known as arcuate fibres, are restricted to a single hemisphere and are subdivided into two major categories, short arcuate fibres and long arcuate fibres. They are the axons of pyramidal cells and fusiform neurons. Short arcuate fibres, which connect adjacent gyri, do not usually reach the subcortical white matter of the cerebral cortex; most of them are confined to the cortical gray matter. The long arcuate fibres, which connect nonadjacent gyri, consist of the following fibre tracts the uncinate fasciculus, cingulum, superior longitudinal fasciculus, inferior longitudinal fasciculus, and fronto-occipital fasciculus (Patestas and Gartner, 2013).

2.9.2 THE HIPPOCAMPUS The hippocampus (plate 2.5a and 2.5 b) phylogenetically is one of the oldest parts of the brain, is a part of the limbic system and a major part of the vertebrates brain, most mammals have two hippocampi, one on each side of the brain. The hippocampus is located beneath the cerebral cortex (Wright, 2014) and in primates it is located in the medial temporal lobe, underneath the cortical surface. It contains two main interlocking parts: Cornu Ammonis (hippocampus proper) and the dentate gyrus (Amaral and Lavenex, 2006). Grossly the hippocampus is a club-shaped structure divided into three parts: head, body, and tail. The head is anterior and is called the pes hippocampus, the body is cylindrical in shape, and the tail tapers posteriorly (Mark et al., 1993). The hippocampus proper is visible on its dorsal aspect, while the dentate gyrus is buried inside. The fimbria is a large fibre tract which is visible on the lateral edge of the exposed hippocampus (Okeefe and Nadal, 1978). The hippocampus has been studied extensively as part of a brain system responsible for spatial memory and navigation in rodents. Many neurons in the rat and mouse hippocampus respond as place cells: that is, they fire bursts of action potentials when the animal passes through a specific part of its environment. Hippocampal place cells interact extensively with head direction cells, whose activity acts as an inertial compass, and conjecturally 43

with grid cells in the neighbouring entorhinal cortex (Amaral and Lavenex, 2006). Different neuronal cell types are neatly organized into layers in the hippocampus; it has frequently been used as a model system for studying neurophysiology. A form of neural plasticity known as longterm potentiation (LTP) was first discovered to occur in the hippocampus and has often been studied in this structure. LTP is widely believed to be one of the main neural mechanisms by which memory is stored in the brain (Amaral and Lavenex, 2006).

2.9.2.1 Histology of the Hippocampus The histology of the hippocampus has been studied extensively since the time of Cajal in 1911. Cajal and Lorente de Nó in 1934 modified the Golgi staining method to improve its effectiveness and employed it in the study of cellular architecture of the hippocampal formation in rodents (Martin et al., 2002). By the middle of the 20th century the hippocampus had become one of the most studied structures of the nervous system. The hippocampus can be viewed as a primitive form of three-layered cortical tissue or a simple cortex, consisting primarily of one basic cell type and its associated interneurones. These basic neurons are packed together in one layer of a three-layered structure, in contrast to the six layers of neocortex (Okeefe and Nadal, 1978; Ahmed and Nameer, 2009; Amin et al., 2013). The gray matter of the hippocampus is an extension of the subiculum of the parahippo-campal gyrus (Mark et al., 1993). The dentate gyrus has three layers: (1) the granule layer containing densely packed cell bodies of the granule cells; (2) the molecular layer formed by the intertwining apical dendrites of the granule cells and their afferents; (3) the polymorph layer in the hilus of the dentate gyrus which merges with the CA4 field and contains the initial segments of the granule-cell axons as they gather together to form the mossy fibre bundle. It also contains a few types of non-granule cells, the most important of which is the basket cell.

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The hippocampus proper (cornu ammonis areas) are made up of four areas 1) CA4 underlies the dentate gyrus, 2) CA3, 3) a small zone called CA2 and 4) CA1. The cornu ammonis areas are all filled with densely packed pyramidal cells similar to those found in the neocortex. The cornu ammonis although a three-layered structure has been divided into as many as seven layers, each defined by a particular feature of the large pyramidal cells or their afferents (Okeefe and Nadal, 1978). These layers extend from the ventricular surface and are : (1) The alveus which contains axons of the pyramidal cells, directed towards the fimbria or the subiculum; (2) the stratum oriens, a layer between the alveus and the pyramidal cell bodies which contains the basal dendrites of the pyramidal cells and some of the basket cells, as well as afferents from the septum; (3) the stratum pyramidale, or pyramidal layer, which is dominated by the cell bodies of the pyramids; (4) the stratum radiatum and (5) the stratum lacunosum/molecular; these last two layers contain apical dendrites of the pyramidal cells and hippocampal afferents from the entorhinal cortex.

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Plate 2.5 a: Drawing of the left rodent hippocampus (Okeefe and Nadal, 1978)

Plate 2.5b: Hippocampal anatomy and internal circuitry. Diagram showing the histological appearance of the cell layers within the hippocampus and loci of the hippocampal fields, dentate gyrus, and subicular cortex. (Adapted from what.when.how.com).

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2.9.3 THE CEREBELLUM The cerebellum (Plate 2.6) is located in the posterior aspect of the brain, just below the occipital lobes of the cerebrum. It is separated from the cerebrum via a horizontal dural reflection, the tentorium cerebelli. The cerebellum is connected to the midbrain, pons, and medulla of the brainstem via three pairs of fibre bundles, the superior, middle, and inferior cerebellar peduncles, respectively. Viewing the cerebellum, it can be seen that it is composed of the right and left cerebellar hemispheres and the narrow, intervening vermis (Patestas and Gartner, 2013). The vermis is also subdivided into a superior and an inferior portion, where the superior portion is visible between the two hemispheres, while its inferior portion is buried between the two hemispheres. The surface of the cerebellum has horizontal elevations, known as folia, and indentations between the folia, known as sulci. Some of these sulci are deeper than others and they are said to subdivide each hemisphere into three lobes, the small anterior lobe, the much larger posterior lobe, and the inferiorly positioned flocculonodular lobe (formed from the nodule of the vermis and the flocculus of each cerebellar hemisphere). The anterior lobe is separated from the posterior lobe by the primary fissure, and the posterolateral fissure separates the flocculonodular lobe from the posterior lobe (Patestas and Gartner, 2013). Similar to the cerebrum, the cerebellum has an outer rim of gray matter, the cortex, an inner core of nerve fibres, the medullary white matter, and the deep cerebellar nuclei, located within the white matter. The cortex and white matter are easily distinguished from each other in a midsagittal section of the cerebellum, where the white matter arborizes, forming the core of what appears to be a tree-like architecture, known as the arbor vitae.

2.9.3.1 Histology of the Cerebellum The cerebellar cortex is a three-layered structure, the outermost molecular layer, the middle Purkinje layer, and the innermost granular layer. The granular layer is well defined due to the presence of nucleic acids in the nuclei of its numerous, small cells. The Purkinje layer, 47

composed of a single layer of large Purkinje cell perikaryons, is also easily recognizable. The molecular layer is rich in axons and dendrites as well as capillaries that penetrate deep into this layer (Patestas and Gartner, 2013). Four pairs of nuclei are located within the substance of the cerebellar white matter. These are the fastigial, dentate, emboliform, and globose nuclei. The connections between the cortical regions and the deep nuclei of the cerebellum permit the subdivision of the cerebellum into three zones, the vermal, paravermal, and hemispheric where each zone is composed of deep cerebellar nuclei, white matter, and cortex (Patestas and Gartner, 2013).

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Plate 2.6 Structural features of the cerebellum (©Pearson’s Education, Inc.)

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CHAPTER THREE 3.0

MATERIALS AND METHODS

3.1

EQUIPMENT AND APPARATUS Open field box, Y maze, Elevated plus maze, Laboratory incubator, Centrifuge, Teflon-

glass tissue homogenizer, UV-Vis spectrophotometer. 3.2

REAGENTS AND DRUGS Aspartame 99.9% purity NutraSweet®

3.3

ANIMALS: Adult male albino mice were used for the experiments. The animals were obtained from

Empire farms in Osogbo, Osun state, Nigeria. They were kept in well ventilated room, fed with standard chow (Premier Feeds, Ibadan, Oyo State, Nigeria) and allowed water ad libitum. The mice received humane care according to the criteria outlined in the “Guidelines for the Use of Animals in Neuroscience and Behavioural Research” prepared by the Committee on Guidelines for the Use of Animals in Neuroscience and Behavioural Research; National Research council of the National Academies (2003).

3.4

EXPERIMENTAL METHOD Sixty adult male Swiss albino mice weighing between 20-22 g were used for the study.

The mice were randomly assigned into 5 groups of 12 animals each. Mice were administered vehicle (distilled water) one of four selected doses of aspartame (20, 40, 80 and 160 mg/kg body weight) daily for 28 days orally. Doses of aspartame were calculated by dissolving measured quantities of the sweetener (Nutrasweet®) in distilled water. Neurobehavioural studies were carried out using standard behavioural tests after the first and last dose of aspartame. The Open field was used to study novelty induced behaviours such as locomotion, rearing and grooming, Y maze for spatial memory and Elevated Plus maze for anxiety. All behavioural tests were recorded using a digital video camera and later scored by two independent observers. At the end of the 50

experimental period, animals were sacrificed by cervical dislocation. Brains were excised and fixed in 10% neutral buffered formalin. Sections of the cerebrum, hippocampus and cerebellum were processed for routine paraffin embedding, cut at 5 µm and stained using hematoxylin and eosin for general histological study, cresyl violet staining protocol was used to demonstrate Nissl substance and Bielschwosky’s silver staining method was used to assess the formation of neuritic plaques and neurodegeneration. Immunohistochemical studies for GFAP and NSE were performed using the Novocastra™ NovoLink™ Polymer Detection System and monoclonal antibodies to GFAP and NSE. All sections were examined using an Olympus trinocular microscope (XSZ107E, Japan) with a digital camera (Canon Powershot 2500) attached and photomicrographs taken. Image J, a National Institute of Health (NIH), USA sponsored software was used for image analysis. Aspartate, superoxide dismutase and nitric oxide levels were assayed from brain homogenates and plasma at 450, 490 and 540 nm absorption respectively with a UV-Vis spectrophotometer (752N, Unigold, England) to ascertain the effect of aspartame at the doses given on brain levels of aspartic acid, and markers of oxidative stress (superoxide dismutase and nitric oxide).

3.5

DETERMINATION OF FOOD, WATER CONSUMPTION AND BODY WEIGHT Animals were kept in single cages and graduated water bottles were placed on the cages,

food hoppers containing a pre-weighed amount of food were provided, and plastic transparency was placed underneath the cages to catch any food spillage. Total food consumption was measured using the difference between the preweighed standard chow and the weight of chow daily. Crumbs in the plastic transparencies were weighed and accounted for in the measurement of total food consumed during the 24-hr period, while total water intake was calculated by weighing the water bottles at the start and end of the 24-hr period. Body weight was measured by weighing animals weekly using a Mettler weighing balance (Mettler Toledo Type BD6000, Greifensee, Switzerland)

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3.6

EXPERIMENTAL MODEL

3.6.1 NOVELTY INDUCED BEHAVIOUR Twenty minutes period of the following behavioural states; locomotion, rearing and grooming, was observed and scored, these was used to characterize behavioural changes in the mice when placed in the open field. The structure was a rectangular box composed of a hard board floor measuring 36x36x26cm and made of white painted wood. The floor was divided by permanent red markings into 16 equal squares at the bottom. Spontaneous motor activity was monitored for 20 minutes in the open field as described by Ajayi and Ukponmwan (1994). Following the first and last doses of either drug or vehicle each mouse was introduced into the field and the total locomotion (number of floor units entered with all paws), rearing frequency (number of times the animal stood on its hind legs or with its fore arms against the walls of the observation cage or free in the air) and frequency of grooming (number of body cleaning with paws, picking of the body and pubis with mouth and face washing actions) within each 10 minute interval recorded. The arena was cleaned with 20 % alcohol to eliminate olfactory bias and allowed to dry before introducing a fresh animal. At the beginning of the test, each animal was placed in the apparatus and its behaviour videotaped for subsequent analysis, behavioural testing was done between 7.00 am and 3.00 pm.

3.6.2 LEARNING AND MEMORY TESTS: Y-MAZE Spontaneous alternation is a measure of spatial working memory; the Y-maze can be used as a measure of short term memory, general locomotor activity and stereotypic behaviour (Olton and Samuelson, 1976). Therefore, spontaneous alternation was assessed using a Y- maze composed of three equally spaced arms (120°, 41cm long and 15cm high). The floor of each arm (5cm wide) was made of wood. Each mouse was placed in one of the arm compartments and allowed to move freely until its tail completely entered another arm. The sequence of arm entries was recorded, the arms being labeled A, B, or C. An alternation is defined as entry into all three 52

arms consecutively, for instance if the animal makes the following arm entries; ACB,CA,B,C,A,CAB,C,A, in this example, the animal made 13 arm entries 8 of which are correct alternations. The number of maximum spontaneous alternations is then the total number of arms entered minus two, and the percentage alternation is calculated as {(actual alternations /maximum alternations) x 100}. For each animal the Y-maze testing was for a period of 5 minutes. The apparatus was cleaned with 20 % alcohol and allowed to dry between sessions (Dellu et al., 1992).

3.6.3 ANXIETY MODEL: ELEVATED PLUS MAZE The elevated plus maze is a plus-shaped apparatus with four arms at right angles to each other as described by Handley and Mithani (1984). The two open arms lie across from each other measuring 25 x 5 x5 cm and perpendicular to two closed arms measuring 25 x 5 x 16 cm with a center platform (5 x 5 x 0.5 cm). The closed arms have a high wall (16 cm) to enclose the arms whereas the open arms have no side wall. Following administration of drug or vehicle, mice were placed in the central platform facing the closed arm and their behaviour recorded for 5 min. based upon studies by Montgomery (Montgomery, 1958; Pellow and File, 1997). The criterion for arm visit was considered only when the animal decisively moved all its four limbs into an arm. The maze was cleaned with 20 % ethanol after each trial. The elevated plus maze relies upon rodents' proclivity toward dark, enclosed spaces (approach) and an unconditioned fear of heights/open spaces (avoidance) (Pellow and File, 1997).The percentage of time spent in the arms was calculated as time in open arms or closed arm/total time x100, the number of entries into the arms was calculated using number of entries into open or closed arms/total number of entries (Barnett, 1975; Hogg, 1996).

3.7

SACRIFICE OF ANIMALS At the end of the experimental period, mice were observed for changes in their physical

characteristics. Sacrifice was by cervical dislocation and the brain of each of the animals dissected 53

out. The brain was observed grossly and then fixed in 10 % neutral buffered formalin for histological studies. Paraffin sections were cut and stained with haematoxylin and eosin for general histological study, cresyl violet staining protocol to demonstrate Nissl substance, Bielschwosky’s silver staining method was used to determine the formation of neuritic plaques and neurodegeneration; and immunohistochemical studies for GFAP and NSE.

3.8

HISTOLOGICAL PROCEDURE Sections of the cerebrum, cerebellum and hippocampus was fixed in 10 % neutral buffered

formalin by total immersion for 24 hours after which they were trimmed to about 3-5mm thick sections and processed via paraffin wax embedding method of Drury and Wallington (1980). The tissues were dehydrated at room temperature through ascending grades of ethanol. Dehydrated tissue was cleared at room temperature in two changes of xylene each lasting one hour. The tissues were then infiltrated in two changes of molten paraffin wax at 60 °C for one hour each and then finally embedded in paraffin wax using a multi block plastic embedding mould. The paraffin blocked tissues were trimmed and mounted for sectioning on a rotary microtome. Sections of 5ųm thickness were produced from the tissue blocks using a rotary microtome (Bright B5143, Huntington, England) the sections were transferred into a water bath (40°C) to allow spreading of the folded ribbons of sections. These sections were mounted on new, clean glass slides. They were then dried to 40°C on a slide drier to enhance adherence of the section to the slides.

3.9

STAINING PROTOCOL Paraffin wax is poorly permeable to stains so sections were deparaffinised in two changes

of xylene for two minutes each. Xylene is removed because it does not mix with aqueous solution and low grade of alcohol used in preparing stains, thus sections were passed through two changes of absolute alcohol for four minutes each. The sections were then hydrated using a series of

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descending concentrations of alcohol until water was used, this allowed for staining with an aqueous dye 3.9.1 HAEMATOXYLIN AND EOSIN STAINING PROTOCOL (H&E) H&E staining was carried out as described by Drury and Wallington (1980). Deparaffinized tissue was rehydrated in descending concentrations of alcohol for 2 minutes each, washed in distilled water three times, and stained with haematoxylin for 20 minutes, and then rinsed with running tap water for 5 minutes. They were subsequently differentiated in 1 % acid alcohol for 10 seconds, rinsed adequately under running water to remove excess acid. The nuclear staining was followed by counterstaining with eosin for 2 minutes, dehydrated through 95 % alcohol, cleared in xylene and mounted on dibutyl phathalate xylene (DPX) medium for microscopic examination.

3.9.2 CRESYL VIOLET STAINING PROTOCOL Cresyl Violet solution was used to stain Nissl substance in the cytoplasm of neurons in formalin-fixed tissue. The neuropil would be stained a granular purple-blue. The Cresyl Violet method uses basic aniline dye to stain RNA blue, and is used to highlight important structural features of neurons. The Nissl substance (rough endoplasmic reticulum) appears dark blue due to the staining of ribosomal RNA, giving the cytoplasm a mottled appearance. Individual granules of extra-nuclear RNA are named Nissl granules (ribosomes). DNA present in the nucleus stains a similar colour. Deparaffinized tissue was rehydrated in descending concentrations of alcohol for 2 minutes each, washed in distilled water three times, and stained with 0.1% Cresyl Violet for 10 minutes; sections were rinsed with running tap water for 5 minutes to remove excess stain following which they were washed in 70 % alcohol and differentiated in 1 % acid alcohol for 10 seconds. Excess acid was removed by rinsing adequately under running water. Sections were then dehydrated through

55

ascending grades of ethanol (50 % through to absolute ethanol), cleared in xylene and mounted on dibutyl phathalate xylene (DPX) medium for microscopic examination.

3.9.3 BIELSCHOWSKY SILVER STAINING PROTOCOL (van Eersel et al., 2010) The Bielschowsky silver staining method is used to demonstrate neurofibrillary tangles, nerve fibres and senile plaques in tissue samples. It works on the principle that the nerve fibres are sensitized with a silver solution. Sections are treated with ammoniacal silver, and then reduced to visible metallic silver. Deparaffinized and rehydrated sections were incubated in pre-warmed (37°C) 20% silver nitrate solution for 15 minutes, washed and then placed in ammonium silver nitrate solution at 40°C for a further 30 minutes. Sections were subsequently developed for 1 minute and then transferred to 1% ammonium hydroxide solution for 1 minute to stop the reaction. Sections were then washed in distilled H2O, placed in 5% sodium thiosulphate solution for 5 minutes, washed, cleared and mounted in dibutyl phathalate xylene (DPX) medium. Axons, plaque neurites and tangles stain black, in a yellow to brown background whilst plaque and vascular amyloid generally stain brown to dark brown.

3.10

IMMUNOHISTOCHEMICAL STAINING Immunohistochemical tests for glial fibrillary acid protein (GFAP) and NSE were

performed using the NovocastraTM and NovolinkDM polymer detection system (Leica Biosystems, UK) and appropriate primary monoclonal antibodies. The Novolink polymer detection system (PDS) utilizes an innovative controlled polymerization technology to prepare polymer horseradish peroxidase (HRP) linker antibody conjugates, hence overcoming the problem of non specific staining as seen with the Streptavidin/Biotin detection systems secondary to exogenous biotin. The Novolink and Novocastra sytems allow for identification of small quantities of antigen in sections of formalin-fixed, paraffin- embedded tissue in a sequence of steps. Endogenous peroxidase activity in the tissues are neutralized using the NovocastraTM peroxidase block, followed by the 56

application of the Novocastra protein block which helps reduce non specific binding between primary antibody and polymer. Sections are sequentially incubated in optimally diluted quantities of primary antibody; penetration of the polymer reagents is improved by using Novocastra post primary block. Sections are further incubated with the substrate chromogen 3’3 diaminobenzadine (DAB) prepared from Novocastra DAB chromogen and Novolink DAB substrate buffer (polymer). Reaction with the peroxidase produces a visible brown precipitate at the antigen site. Monoclonal antibodies (Leica Biosystem) were used for the appropriate antigen glial fibrillary acid protein (GFAP) and non specific enolase (NSE).

3.10.1 IMMUNOHISTOCHEMISTRY PROCTOCOL Sections were taken to water; following which antigen retrieval is performed in Novocastra proteinase K solution for 15 minutes and equilibrated in running water. Sections are blocked in NovolinkTM peroxidase block for 15 minutes, and then washed in phosphate buffered saline (PBS). Tissue protein is blocked by immersion in novolink protein block for 15 minutes, and then sections are washed in PBS, incubated for 15 minutes in NovolinkTM post primary block, washed twice in PBS and then incubated for 15 minutes in Novolink

TM

polymer. Treatment with

Novolink TM DAB working solution is carried out 5 minutes, sections washed twice in PBS, rinsed in water, Sections were counter stained with haematoxylin for 2 minutes, rinsed in water, dehydrated and mounted on synthetic resin medium (DPX) using glass cover slips. All antibodies used were diluted by 1/50 using standard antibody diluents and procedure carried out in a humidity chamber.

3.11

BRAIN ASPARTIC ACID, SUPEROXIDE DISMUTASE AND NITRIC OXIDE At the end of the study, mice were sacrificed and decapitated; the brains were immediately

removed, washed with ice-cold saline solution (0.9 % Na-Cl) and weighed. To expose the brain, the tip of curved scissors was inserted into the foramen magnum and a single lateral cut was made 57

into the skull extending forward on the left and right side. With a bone cutter, the dorsal portion of cranium was peeled off, and using a blunt forceps, the brain was dropped onto the ice-cold glass plate, leaving the olfactory bulbs behind. The whole process of removing brain took less than 2 minutes. After removing the brain, it was blotted and chilled. Further dissection was made on icecold glass plate. Whole brain homogenates were prepared in a Teflon-glass mortar using 100 microL of assay buffer and centrifuged separately at between 1200 and 4000 revolutions per minute (RPM) for 10 minutes. The supernatants (whole brain) were used for analyses. Brain homogenates were added to constituted enzyme mix in quartz glass colorimetric plates and the mixture was incubated (DNP 9022A, Royalcare, England) at 37 degree Celsius for 30 minutes as recommended. A direct reading of concentration was done using a UV Vis spectrophotometer (752 N, Unigold, England) in the C (concentration) mode after instrument calibration. The assay for aspartic acid assay was performed using the aspartic assay kit manufactured by BioVision Incorporated, CA, USA. (Kit batch number CAT.K552-100, Lot number 9G 160629). Aspartate is converted to pyruvate which is oxidized with the conversion of a probe into a highly colored (570 nm) species proportional to the amount of aspartate in samples, the assay was carried out according to the manufacturer’s instructions. The assay for nitric oxide was carried according to the spectrophotometric method of Montgomery and Dymock (1961) in which the resulting azo dye has a bright reddish purple colour and absorbance was measured at 540 nm. Superoxide dismutase levels was assayed according to the method by Nishikimi et al., (1972) based on the ability of the enzyme to inhibit the phenazine methosulphate mediated reduction of nitro blue tetrazolium dye, the change in absorbance at 560 nm over 5 min was measured.

3.12

PHOTOMICROGRAPHY Brain sections were examined under a Sellon Olympus trinocular microscope (XSZ-107 E,

Japan) with a digital camera (Canon powershot 2500) attached.

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3.13

IMAGE J MORPHOMETRIC ANALYSIS To quantify the cell count, % area covered by cell and colour intensity within the cerebral

cortex, hippocampus and cerebellar cortex, an integrated morphology analysis was undertaken using the image J National institute of health software. For each section measurements were conducted on at least 3 representative slides from at least 4 animals per experimental group. Digital brightfield images were uploaded unto the image J analysis software and scale was set using a digital micrcrometer gauge reading to convert measurements in pixels to microns and this was applied globally to all images. Cells were counted using the cell counter plug-in available on the image J analysis software after a grid had been applied across the image; number of the different cell types in the respective brain regions was then counted. Total number of cells, % area covered by cells and average cell size were arrived at using the automated cell counter after thresholding for colour.

3.14

STATISTICAL ANALYSIS

All data were analyzed using analysis of variance (ANOVA), one way ANOVA was used to determine the effect of dose alone (mg/kg body weight) against vehicle in the weight and neurochemistry data, while two factor ANOVA was used to determine the main effect of dose and duration of administration (acute vs. subchronic) in the neurobehavioural tests, Tukey’s multiple comparison post-hoc tests, Tukey highly significant difference (Tukey HSD) was used to identify source of a significant effect. Differences in means of acute vs. subchronic comparisons following administration of aspartame was further compared against difference seen between acute and subchronic following administration of vehicle and only considered significant if effect seen with aspartame differed significantly from vehicle baseline [Aspartame (Acute-subchronic) > vehicle (acute-subchronic)]. Results were expressed as Mean ± S.E.M., p