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The Effect of Choline on Spatial Memory of Age-Impaired and Non-Age-Impaired Rats Essays

Abstract
Choline has been identified as one of the essential nutrients of the human body. Choline has served many different functions to the human body. Choline is also said to have an effect on memory performance. In this paper, the researcher investigates the effect of choline in rats’ memory performance. The research also investigates the effects of choline in rats with age impairment. The researcher collects a total of 64 rats sample in order to conduct an experiment. From the 64 sample, 32 are age impaired and 32 are non-age impaired. The rats are further grouped to non-choline and choline administered rats. The performances of the different groups are determined using Morris maze experiment.
A 2 x 2 factorial analysis of variance was conducted in order to determine significant effects of choline. After conducting the test, the researcher found out that choline has a significant effect on the memory performance of rats [F (1,28) = 12.00, p < .01]. On the other hand, the researcher found out that age impairment [F (1,28) = 0.015, p > .05] and the interaction between age impairment and choline administration [F (1,28) = 0.075, p > .05] do not have a significant effect on the memory performance of rats.

The Effect of Choline on Spatial Memory of Age-Impaired and Non-Age-Impaired Rats
The National Academy of Sciences identified choline as an essential nutrient for humans in 1998 (Institute of Medicine and National Academy of Sciences, 1998). Choline is the  chemical building block used as a component of the phospholipids, phosphatidylcholine and sphingomyelin. Smaller amounts are needed to generate the neurotransmitter acetylcholine, platelet-activating factor, lysophosphatidylcholine and betaine (Zeisel, 2000).  Choline previously was thought to be an unofficial B vitamin because it is closely linked to vitamins B-6, B-12 and folate through a complicated chemical cycle called the SAM (s-adenosyl-methionine) cycle. The movement of chemical structures around the SAM cycle is dependent on folate. Therefore, folic acid deficiency may potentially undermine the choline status by disrupting the SAM cycle balance (Allman-Farinell et al., 1999) Although the body can make choline, it cannot synthesize enough to maintain proper health and functioning, therefore sufficient dietary intake is critical. The best dietary sources are eggs and liver followed by soybeans, peanuts, potatoes, and lentils (Bhagwat et al., 2008).
Choline’s nutritional value had been studied for almost 150 years; findings showed the pancreas, liver, and every human cell contained choline.  Experiments involving brain health, cognitive behavior, and diseases such as Alzheimer’s disease are just now being conducted.  Other benefits include its vital role in ensuring proper functioning of the liver and its role in the production and metabolism of protein and fat (Bear et al., 2007).  Because of its critical importance to so many metabolic functions and its late discovery as an essential nutrient, I wanted to expand the knowledge about this important nutrient.
Richard Morris designed a study instrument known as the Morris water maze in an attempt to determine different effects of chronic, continuous and acute exercises within differently assigned periods (Matell & Portugal, 2007).  The Morris water maze is accepted worldwide as the most effective method to research the “psychological process and neural mechanisms of spatial learning and memory” (Morris, 2008). The subjects, usually rats, are put in a large circular pool of water. In order to escape from the pool onto the platform, they must use a combination of skills including using the local cues in the room to form a cognitive map. Neurons in the hippocampus (responsible for encoding long-term memories) will indicate points in the spatial area for the rats to find their way out (Morris, 2008).
During the fetal period, important cognitive development occurs. A study performed at Duke University Medical Center gave a pregnant rat choline in their third trimester. The infant rats born to these mothers “had vastly superior brains, improved learning ability, and better memory recall, all of which persisted into old age” (Holford, 2001). Currently, nutritional experts are focusing on choline and later cognitive behaviors (Franklin et al., 2006). It appears that choline is important to all periods of cognitive development. Choline, when given to aged rats or elderly humans, greatly improves brain and memory functions (Franklin et al., 2006).
Normal muscle functioning is also determined by the amount of choline in the body.  It also plays a significant role in humans by reducing sleep problems. Choline assists the brain to shut out minor noises in the surrounding environment, allowing the individual to enjoy a good night’s rest (Cheng & Meck, 2007).
I propose an experiment to test the effects of choline on rats and age-impaired rats concerning memory.  A small number of studies have been implemented to determine the effects of choline on aged rats. Findings revealed that choline is a memory booster in older rats, compared to same age rats not given the nutrient (Nieuwenhuyzen & Szuhaj, 2003).  Choline had no effect on younger rats.  The positive results in the older rats are “attributed primarily to a presumed increase in phosphatidylcholine synthesis in the brain” (Block, 2010).
  According to Matell and Portugal, the conditions of the environment in which rats live affect their cognitive performance (Matell & Portugal, 2007).  The normal functioning of the hippocampus is vital for spatial memory processing. Conflicting experiments found that neither environmental conditions nor aging compromised functional memory. However, conflicting experiments show that neither impoverished conditions nor aging can compromise practical memory (McDowell, 2000).  This theory argues that the stimulus-response memory, also known as ‘habit of the rat’, is independent of the hippocampus (Nix, 2005). The present literature shows a conflict on whether or not environmental conditions do indeed affect rats’ memory. Further study and experimentation need to be conducted in order to resolve this conflict.
The main goal of the present experiment is to identify how rats learn, how they retain information, and how their age affects these processes. Massachusetts Institute of Technology, Department of Brain and Cognitive Sciences, used Morris’s water maze theory to test the memory of rats given choline daily for two to three months before testing started. The rats not given choline continued with their declining memory (Block, 2010) and had the tendency to travel longer distances in order to find the hidden platform in the water maze compared to the other group (Matell & Portugal, 2007). The rats under the choline intervention completed their task of finding the hidden platform in a shorter time  (Bear et al., 2007).
The cognitive deficit in the rats not given the nutrient may have resulted from failure to remember the location of the hidden platform due to their inadequate consolidation of information from impaired memory retrieval mechanisms (Franklin et al., 2006).  A few challenges were encountered while conducting the experiment using choline on rats.  For instance, at the beginning of the experiment, the rats were dropped head first into the pool.  It was discovered later that the rats whose heads were dropped first were so stressed that the learning was impaired (Raven, 2008).  The rats should be lowered gently with their tail end lower to prevent the head going under water.
In the present experiment, a two by two factorial design will be employed to test the effects of choline on age impaired and age non-impaired rats. The water maze procedure will be utilized. There will be four groups for the experiment and they will include age impaired rats given choline, age impaired rats not given choline, age non-impaired rats given choline, and age non-impaired rats not given choline. There will be testing before experimenting begins to determine which of the older rats are age impaired.
Groups not given choline will be given a placebo. The administration of the placebo will be simultaneous to the administration of choline to the experimental groups. Anticipated observation time will be two to three weeks. Observation will involve monitoring each group as they pass through the water maze, interact with other rats, and conduct memory tests.  One such memory test will be providing food in a centralized location, attempting to confuse the rats in the water maze, and seeing which ones successfully reach the area with the food.
It is hypothesized that the older rats given the choline intervention will thrive and that they will have improved memory and faster resolution time. Memory will be measured in terms of successful completion of the Morris maze. Timed trials will be conducted with the Morris water maze to establish the differences in memory between the trial groups. The groups with the aged non-impaired rats are expected to perform poorest. It is also expected that non-age impaired rats will not show significant differences in performance in terms of their choline status.
Methods

In order to ensure accuracy of the results, a total of 64 rats were used in the experiment, all of them were males. This was done in order to eliminate the possible effects of gender on task performance. Moreover, 32 of these rats were age impaired (22-24 months of age) while the other 32 were young (3-4 months of age). Age categories were based on the criteria set by Wang et al. (2003). Initially, the tails of the rats were wrapped using masking tape marked with a clearly visible identifying number code. Clear marking is necessary to facilitate proper recording of results. Consequently, the rats were randomly assigned into four categories:  age impaired and choline-administered rats, age impaired control group, age non-impaired choline-administered rats, and age non-impaired control group. The assignment of groups was done through a list of random numbers in which the experimenter picked a number from the list and the rat with the corresponding number taped in its tail was assigned to a particular group. In this manner, each group consisted of 16 rats in total.
To prepare the subjects for the experiment, all rats were fed with standard laboratory rat pellets and were given free access to water. However, two of the groups that were assigned to receive choline were supplemented with 0.4% choline chloride (Enomoto et al., 1998). The nutrient was administered intraperitonealy (Klein et al., 1992). Likewise, the amount of food given is maintained at a ratio of 1 gram of food per 20 grams of body weight such that a rat weighing 80 grams, for instance, is given 4 grams of food (Chow et al., 2008).  Moreover, just like other experiments conducted on rats (Arias et al., 2004; Enomoto et al., 1998), the subjects were also maintained at a 12-hour light-dark cycle. These dietary conditions were maintained for two weeks prior to the experiment proper.
            The Morris maze experiment apparatus used in the experiment consisted of a custom-made circular white basin which is 150 cm in diameter, with a depth of 40 cm (Arias et al., 2004). The basin was divided into four imaginary quadrants delineated conceptually by the experimenters. Consequently, a round platform was placed in one quadrant at about 25cm from the wall of the basin. The platform was white in color which was the same with that of the basin so that it could not be seen easily by the rats. The basin was then filled with water up to 1 cm above the surface of the platform. Moreover, in order to avoid the effects of external factors, the water temperature was kept within 23ºC – 25 ºC. The pool was then placed in a table located at the center of a room. The room was approximately 300 cm by 500 cm in size, which is neither too crowded nor to wide, providing just enough space for the table and the pool and for the rats to see the cues posted on the walls.
The experiment made use of cued Morris water maze, that is, several cues were placed in different locations around the pool that would aid the rats in learning the spatial position of the platform relative to its environment. Four triangular paper flags, that are black and white, were placed in each corner of the room. Also, colorful huge posters were posted in each wall of the room. In addition, four volunteer experimenters holding a time recorder also served as cues, each one of them was asked to stand in each of the quadrants. They were also instructed to wear white laboratory gown over their clothes all the time in order to maintain the conditions constant.
The experiment was held from 9:00 am to 11:00 am. The procedure began by placing each rat in the Morris maze. Each rat from each group was dropped at a time until all 64 rats were observed. Each volunteer experimenter standing in each of the quadrants was responsible in placing each rat in its corresponding quadrant, taking note of the distance of each rat from the hidden platform. Moreover, the manner by which the rats were dropped was done by holding the body and slowly placing the hind part first in the pool of water before the head. This is because it was found out that rats whose heads were dropped first tend to experience stress that may later impair learning (Raven, 2008), thereby affecting the results.
  The volunteers were not only responsible for dropping the rats but also for time keeping. After placing the subjects in the basin, the rats were first given 20 seconds to explore the entire area. After 20 seconds, each volunteer started the timer and waited until the rat was able to find the platform and stay there for five seconds. This was done to ensure that the rat had indeed found the platform and had not reached the area merely by accident. After five seconds, the rat was removed from the basin and the time was recorded. The cut-off time for a trial is 120 seconds (Holscher et al., 1996). Hence, those rats that failed to locate the platform after such time were manually placed in the platform for 10 seconds until they eventually learned the location of the platform (Holscher et al., 1996).
The volunteer experimenters also took note of any significant behaviors exhibited by the rats that were either removed from or were not placed in the pool yet. In accordance to the guidelines drafted by the NSW Animal Research Review Panel, these rats were housed in transparent plastic tubs that would let them avoid light and outside activities that could distract or upset them. Moreover, based on the same guidelines issued by NSW Animal Research Review Panel, the sizes of these tubs were varied depending on the age of the rats so that only 12 young ones were housed in a cage of 2,000 cm2 of floor area, while the old ones were housed in tubs which are 22 cm high.   However, rats belonging to different groups were not placed in the same tubs. Meanwhile, examples of behavior that were noted include interaction with other rats, the time it takes for a rat to stay in one position, speed of movement, etc. This was to ensure that the rats were able to carry out normal behaviors in compliance to the aforementioned guidelines which are aimed towards maintaining the well-being of animals being studied.
The procedure was repeated every day for one week to come up with different trials. This was to give the rats sufficient time to rest and to learn the spatial relationships that surround them. Another reason for doing only one trial in a day was to prevent the rat from getting used to the water that might, in turn, lessen their desire to escape. Moreover, for each trial, each rat was placed at a quadrant and at a distance from the platform which was different from where they were previously placed. The platform, on other hand, was retained in the same position. In this manner, the rats were able to familiarize themselves with the cues and use these in order to find the hidden platform. The results of all these trials for one week were recorded and plotted in a chart where it would be easy to see the effects of administering choline on both age impaired and non-age impaired rats.
In order to confirm the results of the experiment, another set of tests was done on the following week in which the cues that were previously present in the experiment were varied. The triangular paper flags were replaced with rectangular ones. Further, the posters in the walls of the rooms were changed in position and the volunteer experimenters were then required to wear blue scrub suits over their clothes.  In addition, the location of the platform was also changed such that it was then placed 50 cm from the wall of the basin. This new setup was maintained for another one week. This was done to confuse the rats and to test whether they would learn how to reach the platform again. The same procedures of recording the behavior, recording the time it takes for the rat to find the hidden platform and plotting it in a graph were repeated. All the results gathered were then compared for analysis.
In general, the series of experiments lasted for two weeks after the diet was started. Throughout this span of time, the same specialized diets were given to each group of rats. However, the amount of food was changed in order to maintain the ratio of 4 grams of food for every 80 grams of the rat’s body weight  (Chow et al., 2008). This was done because the young rats were growing in size significantly during the entire month of the experiment.
Results
A 2 x 2 (Choline x Age) factorial analysis of variance (ANOVA) was conducted for the dependent measure, escape latency.  The results showed a significant main effect of Choline [F (1,28) = 12.00, p < .01]. However, the results also showed insignificant main effect of Age [F (1,28) = 0.015, p > .05] and an insignificant Choline x Age interaction [F (1,28) = 0.075, p > .05].
As seen in Figure 1, rats that are choline administered successfully finished the maze in a significantly faster time than rats that are non-choline administered. On the other hand, one can see that the latency of both age and non-age impaired rats does not differ significantly in choline and non-choline administered states.
Discussion
The results showed evidence to support that there is a significant difference in the latency of rats in finishing the race between non-choline and choline administered rats. While the effects of choline have been found to be significant to rats, the researcher however failed to find evidence to show that there is a significant difference in latency between age and non-age impaired rats. Based on the literature review, choline has been proven important in different periods of cognitive development (Franklin et al., 2006). Choline, when given to non-age and age impaired rats, give the same result of decreased time in finishing the maze.
The researcher found a number of limitations regarding the study. The first limitation observed from the study is the use of male rats for the experiment. Although the researcher wanted to eliminate the effects of gender on task performance, the researcher created another problem, which is gender bias. Another limitation obtained from the study is the amount of choline used for the experiment. The amount of choline administered to patients was 0.4% (Enomoto et al., 1998). Although the amount has already given a significant effect, the researcher may have obtained a more significant effect when the amount of choline administered was increased to a certain level.
Several suggestions for further research are obtained by the researcher. First, the researcher suggested that one should also look for the possible effects of choline across gender of rats. The suggestion is created in order to avoid gender bias and to study the effects of choline in the task performance in terms of gender. Another suggestion of the researcher is to study the effects of increased amount of choline to a rat’s memory. The effects of increased amount of choline administered to a rat may have an increased effect on the task performance of rats.
In conclusion, both the results and the literature have given evidence to say that choline has a significant effect on the memory performance of rats. However, age impairment and the interaction between age impairment and choline administration do not seem to have an effect on the memory performance of rats.
References
Allman-Farinell et al. (1999) ‘Folate nutriture alters choline status of women and men fed low choline diets’. Journal of Nutrition, 129, 712-717.
Animal Research Review Panel (n.d.) ‘Guidelines for the housing of rats in scientific institutions’. Retrieved April 16, 2010 from <http://www.animalethics.org.au/__data/assets/pdf_file/0014/222512/housing-rats-scientific-institutions.pdf>.
Arias, J. et al. (2004) ‘Sex differences in the Morris water maze in young rats: Temporal dimensions’. Psicotherma, 16(4).
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