Betaine and Alcohol Exposure

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Introduction Prenatal exposure to alcohol can bring about numerous adverse outcomes. It is the most common preventable cause of birth defects(xxxxn add reference). By disrupting natural brain development, prenatal alcohol exposure can lead to serious central nervous system (CNS) dysfunctions including behavioral impairments (See Kodituwakku, P.W., 2007, Mattson, S.N., and E.P. Riley 1998 for review). However, regardless of the known consequences that alcohol may have on the developing fetus, pregnant women around the world continue to drink alcohol (Tsai, J., et al., 2009). Children exposed to alcohol during gestation may exhibit facial dysmorphology (Hoyme, H.E., et al., 2005), pre- and postnatal growth deficiencies (Clarren, S.K., et al., 2001), and neuropathology in brain areas including the cerebellum, hippocampus and cortex (Kelly, Day, & Streissguth, 2000). These wide range of outcomes falls under the umbrella term of fetal alcohol spectrum disorders (FASD). Alcohol-induced neuropathology can lead to learning impairments, attention deficits, motor dysfunction, and atypical social conduct (xxxx add example) (Riley, & McGee, 2005). Individuals with FASD suffer from many physical, social, emotional, and cognitive problems, which affect their daily functioning and have life-long implications (2005 xxxx check this out).

	Outcomes among FASD children vary widely.  Since higher rates of FASD are observed in countries where malnutrition is prevalent, this variability may be due, at least partially, to nutritional factors (May, Brooke, Gossage, et al., 2000). Considering the incredible impact of FASD on both individuals and society as a whole, developing efficacious and effective treatment programs that improve behavioral outcome of individuals with FASD is crucial. Furthermore, there is a need for interventions that will help lower the severity of the neuropathology observed in alcohol-exposed children.
	Using an animal model, previous studies in our lab have shown that nutritional choline supplementation can reduce the severity of alcohol’s teratogenic effects, whether administered during alcohol exposure or after alcohol exposure has ended (Thomas, J.D., E.J. Abou, and H.D. Dominguez, 2009, Thomas, J.D., et al, 2007, Thomas, J.D., et al, 2000). Choline is an essential nutrient critical for normal brain development (Food and Nutrition Board, 1998). An essential nutrient describes a nutrient that the body requires in order to function at a normal and healthy level. In other words, such nutrients are not produced within the body at a significant level and thus, must be obtained through dietary ingestion. Choline can be found in a variety of foods such as egg yolks, beef, chicken, and soy (Nutrition Board 1998). Choline is a key component in the homocysteine methionine cycle and involved in various cellular processes Fig. 1. It serves as a precursor to acetylcholine (a neurotransmitter involved in peripheral and central nervous system functions); it helps stabilize cell 


Fig. 1 Homocysteine Methionine Cycle. First, Choline is a precursor to acetylcholine which is the neurotransmitter associated with learning and attention (add reference). Second, Choline serves as a precursor to phosphatidylcholine (a key cellular membrane component) and as a precursor to Betaine. Third, choline can act as a methyl donor, thereby having the ability to regulate gene expression.

membranes, and works as a methyl donor (Zeisel, & Blusztajn, 1994; Meck, & Williams, 2003). My study investigated whether choline’s mitigative effects on deficits resulting from developmental ethanol exposure stems from this last path of methylation, by looking at betaine.

	Betaine, also known as Trimethylglycine (TMG) decreases the amount of homocysteine in the blood; which reduces the risk of cardiovascular disease, and stimulates the creation of adenosine triphosphate (ATP) - our body’s main energy molecule (Zeisel, S.H., 2006). Betaine can be found at high levels in certain foods and absorbed directly by the body, or it can be the product of the body’s oxidization of choline (Zeisel, S.H 2006). Similar to choline, Betaine acts as a methyl donor, affecting DNA methylation and thus, gene expression(xxxx add reference).. Previous studies have shown that betaine may act as a protective agent against cytotoxic brain damage induced by chronic ethanol exposure (Liu, Y., et al., 2009 xxxxxx elaborate as to how it was neuroprotective). The purpose of the present study was to investigate if Betaine could also reduce the adverse consequences of alcohol consumption during pregnancy, and if so, determine the optimal levels necessary to see such effects.

Methods Subjects

	Before mating, each dam was housed in a single cage in a temperature and humidity controlled room, and had ad-lib access to food and water. When we decided it was time for the Dams to mate, they were each housed in a very small cage with male rats until presence of a seminal plug was found, indicating mating, and was designated as gestational day (GD) 0. However, false positives and false negatives could happen, in which case the dams (GD) 0 would have to be estimated by the person whose daily task was to check for the presence of a seminal plug.
	After 22 days of gestation, the offspring would be born. Upon birth, rats would be designated as being postnatal day PD (0). If rats were born one day early, then PD (0) would be designated on the day they were supposed to be born (on their actual PD 1).  On PD 2, rats would be culled to 8 pups (4 males, 4 females whenever possible). The procedure for culling the pups required the experimenter to use scissors in order to cut the underdeveloped heads off the pups and to discard of the animals in biohazardous waste bags.  On PD 6, India ink was injected into the subjects’ paws for future identification.  Each paw in the rat sympolized a pre-determined number. A tattoo on the left forelimb was designated as the number 2, a tattoo on the right forelimb represented number 1, on the left hindlimb represented number 8, and on the right hindlimb represented number 4. With these numbers, rats could be numbered from 1 to 15 (for example: a rat whose number was 9, would be tattooed on the right forelimb and on the left hindlimb). Body weights were recorded from PD4-30, and at the beginning of each behavioral task. From PD4-9, body weights were recorded before the intubation procedure. From PD9 – 30, bodyweights were recorded before the injection procedure. 
	The number of total subjects varied by behavioral task. Some of the behavioral tasks presented in my thesis were discontinued since the task showed no ethanol effect, and it was decided that the test was no longer necessary to continue. Thus, for the Beam task there was a total of (xxxxx) rats. For the activity task, there was a total of (xxxx) rats, and for the odor and object task, there was a total of (xxxxxxxx) rats. 

Treatments

	On PD4 (the first day of intubations,) pups were randomly assigned to one of six treatments groups. Four of them were exposed to alcohol, and the other two were not. The ethanol-exposed rats were treated with a total of 5.25 g/kg ethanol every day from PD 4 through PD 9 via 2 ingragastric intubations. Each intubation was given 2 hrs apart  and was followed by 2 milk feedings with a 2 hour interval in between. The reason behind the milk feedings was because Ethanol-exposed pups tend to be inactive and unable to suckle normally from the dam. Therefore, to reduce the risk of weight loss rats were giving two milk feedings after receiving to alcohol intubations. In terms of neurological development in relation to birth, rats differ from humans. Their brain growth spurt occurs after birth; when in humans the brain growth spurt occurs during the third trimester of pregnancy. Furthermore, the brain growth spurt was of particular importance because it is a critical period of rapid development for the Central Nervous system, and a time of particular vulnerability when exposed to a teratogen like alcohol. Therefore, we exposed the rats to alcohol during this time, for six days during their third trimester equivalent (or postnatal day 4-9).  Controls received sham intubations, with no milk formula. During the alcohol treatment as well as for a period afterwards, we injected these rats with betaine from PD4-30 in order to maximize its effects. For the period when rats received ethanol intubations, I injected the rats with Betaine, immediately following the 2nd intubation. Following this period, Chris injected the rats from PD9-PD30. Three of the ethanol-exposed groups and one of the sham control groups were injected with Betaine, and the other groups (ethanol-exposed and control) were injected with vehicle (saline). Three doses of Betaine were used: 100, 300, and 500 mg/kg at a dose volume of 10 ml/kg. The doses given were based on recommended dosages for pediatric patients with elevated homocysteine blood levels (Matthews, et al., 2002) and based on similar doses we used in previous choline studies (add reference xxxxxx). 

Making Ethanol Diet

	To make an 11.9 % volume per volume solution, I added 7.16 mL of 95 % ethanol to 50 mL of diet. To do so, I used a 50 mL container of milk from one of the freezers in our lab. I let it sit in warm water until it defrosted and then poured the 7.16 mL of 95% ethanol using 6ml and 1ml syringes. The volume of ethanol intubation given to each subject was determined using the subject subject’s body weight. The body weight was multiplied by .33 and divided by 12. 

Making Betaine Solution

	To make the 100 mg/kg concentration of betaine, I added 5g of betaine to 50 ml of double distilled sterile water, and placed the container in an ultrasonic machine until the solution completely dissolved. To make the 300 mg/kg and 500 mg/kg concentrations of betaine, the same process was repeated except 15g and 25g of betaine were added respectively. 
Intragastric Intubation Procedure 
	A length of flexible tubing (Intramedic; Clay Adams Brand; USA) attached to a 25-gauge needle on a 1 mL syringe was used to intubate each neonatal rat. Before the intubation procedure, the distance from the mouth to the stomach was first measured externally and marked, in order to make sure the correct length of tubing was inserted and prevent an incorrect and fatal intubation. The next step was to lubricate the tubing with corn oil, and then to insert the tubing over the tongue and into the stomach. The milk solution containing alcohol was delivered over a minimum of 10 seconds in order to reduce excessive pressure or force, and thus reducing the risk of a fatal or damaging intubation. If I made an incorrect intubation I would know immediately either because I could see milk coming out of the rats nose, or I could see that the rat was truing pale. At this point, I immediately used scissors to cut the head off the rat, and prevent it from further suffering. 
	Ethanol-exposed: As mentioned before, two ethanol feedings were given 2 hrs apart by intragastric intubation at a total dose of 5.25 g/kg, using an 11.9% v/v ethanol in milk solution. The volume of solution delivered to each pup in each feeding was calculated to equal 3 g/kg. Since alcohol exposed pups are less active and are unable to suckle as they normally would, they were given additional milk-only feedings (2 hrs apart) after the ethanol exposure to prevent weight loss.  
	Sham Intubations: Intubation controls have shown higher body weight trends compared to un-intubated or suckle controls (Goodlett and Johnson, 1997; Goodlett et al., 1998). This happens because they are more active and receive additional milk feedings from the dam. For this reason, the sham-intubated animals were intubated in the same way as the ethanol-exposed group, but no milk solution was given. These sham intubations controlled for any potential effects that might have been caused by the intragastric intubation procedure. 

Blood Samples

	 On PD 6, 1.5 hours after the second ethanol intubation, (at the time where blood alcohol concentration would be the highest) 20 micro litters of blood from each subject was collected, by cutting the tip of their tail. The blood samples were analyzed using an Analox Alcohol Analyzer (Model AM1, Analox Instruments; Lunenburg, MA).  The blood samples would tell us the amount of alcohol present in the pup’s blood at the point of highest alcohol concentration, and gave us an estimation of the average alcohol the rat was given.  

Perfusions

	On PD 95, all subjects were perfused and their brains were collected for examination. Since prenatal alcohol exposure imaging studies have shown large reductions in brain areas like the cerebellum and the basal ganglia (Clarren, S.K., et al., 1978), the brains collected will be used for future evaluation of hippocampal, cerebellar and striatal cell loss, further determining Betaine’s effects on the brain. 
Behavioral Testing

Beginning on postnatal day 30 (the last day of Betaine treatments,) rats underwent a series of behavioral tests to evaluate their neuropathology. For this study, I will be presenting activity, parallel bars, elevated beam task and odor & object recognition. I personally tested the rats for the elevated beam task, odor & objects and the first few litters of activity. Chris will be presenting parallel bars, Morris water Maze, visual platform, and working memory. Open field activity

	 From PD 30 to 33, subjects’ activity levels were measured in an automated open field chamber made of Plexiglas (16 in. wide by 18 in. long by 15 in. high [41 cm by 46 cm by 38 cm]). Each open field was housed in a sound-attenuating box containing a fan, which provided ventilation. The open field contained an array of infrared beams that tracked each subject’s movement. Subjects were acclimated to the testing room for 30 minutes before testing. White noise was played during habituation to the room and during the task in order to reduce external noises, which could potentially affect the rat’s behavior.  Before and after testing each box was cleaned to remove any odors that could affect natural activity. To begin testing, each subject was placed in the open field, facing forward and close to the front wall. Once the rat was placed inside the open field, the experimenter would press a button located at the bottom of the chamber, at which point activity began to record. Activity was recorded in 5-min bins for a period of 1 hr per day during the subjects’ dark cycle. Basic movement (the number of times the chamber’s beams break,) the number of center entries, center time, total distance, total time, and center distance served as our dependent variables.  

Parallel Bar Testing

	 On PD 37-39, motor coordination was measured on the parallel bar task. All subjects were tested for 3 consecutive days during the light cycle.  The parallel bar apparatus was made up of two parallel steel rods measuring 0.5 cm in diameter and 91 cm long. They were held between end platforms measuring 15.5 x 17.8 cm. These rods were fastened with screws onto a rack that had 28 slots that were spaced 0.5 cm apart on each platform. The subject was placed on the midpoint between the two platforms.  The animal’s left paws were placed on one rod and right paws were placed on the other. Four successive alternating steps with the hind paws on the rods would be considered a successful trial. 
	The initial distance between rods was set at 3.5 cm.  Subjects were allowed up to five trials at a given width, with an inter-trial interval of 5 to 10 seconds where the rats were placed on the platform where it was headed. If unsuccessful after 5 consecutive trials, testing was terminated for the day.  If the subject placed both hind paws on one rod, fell, dragged a paw, swung under the bars, or turned 360 degrees, the trial was considered unsuccessful and the subject was removed and placed on the platform where it was headed.  If the subject failed to move after a period of 5 minutes, it was placed back in its home cage for one minute and then placed back on the rods.  If the subject failed to move after 3 minutes, testing was terminated for the day and the subject started that day’s testing again on the next day.   Once successful at a given width, the rods were moved at increments of 0.5 cm while the subject remained on the platform.  Subjects were tested up to a maximum of 15 trials per day, for three consecutive days.  Each day the distance between the rods was set to the subject’s last successful width of the previous day. Total width, and the ratio of successful trials to total trials, served as our dependent variables.
	Elevated Beam: On PD 80-82 an elevated beam task was run. The beam-walking apparatus consisted of a wooden beam with ledges underneath each side that allowed for foot faults without having the animal fall from the beam. One platform was located on each side of the beam. The beam was visually divided into three equal parts (having the widest part of the beam on one side, and the least widest in the other end. 
	1. Pre-training:  for 2 days subjects were trained to cross the length of the beam. Fruit loops were used as treats in the start point in order to motivate the rats to cross the beam. The first day the rats were persuaded to cross each section of the beam, starting with the least wide, until three successful traversals were recorded. On the second day of training, they began at the starting point until they successfully completed 5 traversals. 
	2. Test day: For the test day, animals were tested for 5 trials. Two experimenters were needed for this task. While I placed the rat in the starting point and used treats to motivate the animal to cross the beam, my assistant videotaped the rats’ performance. The videos were later analyzed by determining the number of slips. Steps onto the ledge were scored as a full slip and a half slip scored if the subject’s limb touched the side of the beam. Statistical analysis were calculated using the slip ratio (number of slips/number of total steps,) and served as dependent variable.  

Odor and Object Recognition

	 On PD 86-90, an odor and object recognition task was run. This task consisted of three parts: pre-habituation, habituation, and test day procedures.  The pre-habituation phase was used to familiarize the subjects to odors or objects. Habituation was used to habituate the animals to a novel odor or object, and the test day was used to determine the subject’s ability to differentiate between a familiar or novel odor/object.  
	1. Pre-habituation procedures: four 2.5cm round wooden beads per rat were introduced into the subject’s cages. The animals were housed with the beads for 24 hours to allow for familiarization of presence of the beads, as well as for the beads to acquire the odor of the animal and to be used as familiar odors for later use in the experiment. In addition, beads were also introduced into the cages of four odor donors (two male and two female). Male donors were used for male test subjects and female donors for female test subjects.  These beads provided the novel odors necessary for the upcoming habituation and testing procedures. The beads placed on donor odor cages were counterbalanced, so that any one odor could serve as either novel odor 1 (N1) or as novel odor 2 (N2). 
	2. Habituation to N1: after the completion of the 24 hours of familiarization to the beads, the subjects were taken to the testing room and habituated to the environment for 30 minutes before the habituation procedure commenced. After the thirty minutes had elapsed, all the beads were removed from the subjects’ cage. Novel odors from the donor cages were placed in a ziplog bag, sealed and brought to the testing room. The test subjects who were housed together were removed from their cage and placed in another cages sitting next to their cage. Next, a subject would be placed back into his home-cage and exposed to three of the familiar beads and to one of the novel odors taken from the ziplog bag.  There were a total of three 1-minute trials with 1-minute inter-trial intervals (ITI). During the ITI the beads were removed to prevent olfactory adaptation and thus ensuring lasting memory of N1. Whenever the subject approached the bead for the first time, marked the beginning of the one-minute trial.  Exploration time for each of the four beads was recorded using a keypad and (xxxxx software), and the spatial arrangement of the beads in the middle of the cage was randomly altered between trials.  In addition, the N1 bead was discarded after habituation and replaced by another N1 bead taken from the same odor-donor cage in order to remove scent marking as a confounding factor. 
	3. Odor recognition memory assessment:  the odor recognition test was performed 24 hours after the habituation procedure. For this part of the task, rats were presented with two familiar beads, the novel odor N1 they explored the day before, and an unfamiliar novel odor bead (N2) taken from a different odor-donor cage.  For this part of the task, one trial was recorded following the same procedure mentioned in the habituation phase. Figure 2 shows a graphical representation of the experimental procedure. 


Fig. 2 Graphical representation of the three phases of odor recognition task. The Familiar odors correspond to the gray circles, while N1 and N2 correspond to the white and black circles respectively

Object Recognition. Test procedures were identical to that of odor recognition except for the following three things: 1) Odors are substituted for objects (having the round beads serving as the familiar objects, and square and spindle as N1 and N2), 2) donor cages are no longer necessary, 3) odor is removed as a confounding variable by placing the objects in a bag containing a some of the subject’s home cage bedding.


Results Body Weights

	There was a significant difference in body weights as a function of treatment groups (staaaaaaats). The EtOH 500 group had significantly lower body weights than all other groups. The Sham 500 had significantly lower body weights than all other groups except the EtOH 500, Fig 3. 

Activity Levels:

	There were no significant differences in basic movement as a function of treatment group, F(5, 82) = .791, p = .559. There were no significant differences in basic movement as a function of sex, F (1, 82) = .017, p = .896, and there was no interaction between sex and treatment group, F(5, 82) = .238, p = .945. 
	There were no significant differences in center entries as a function of treatment group, F(5, 82) = 1.369, p = .245. There were no significant differences in center entries as a function of sex, F (1, 82) = .337 p = .563, and there was no interaction between sex and treatment group, F(5,65) = .078, p = .995. 
	There were no significant differences in periphery distance traveled as a function of treatment group, F(5, 82) = .791, p = .559. There were no significant differences in periphery distance traveled as a function of sex, F (1, 82) = .017, p = .896, and there was no interaction between sex and treatment group, F(5, 82) = .238, p = .945. 
	There were no significant differences in rearing as a function of treatment group, F(5, 82) = 1.369, p = .245. There were no significant differences in rearing  as a function of sex, F (1, 82) = .337 p = .563, and there was no interaction between sex and treatment group, F(5,65) = .078, p = .995. 
	There were no significant differences in periphery time as a function of treatment group, F(5, 82) = .791, p = .559. There were no significant differences in periphery time as a function of sex, F (1, 82) = .017, p = .896, and there was no interaction between sex and treatment group, F(5, 82) = .238, p = .945. 
	There were no significant differences in center time as a function of treatment group, F(5, 82) = 1.369, p = .245. There were no significant differences in center time as a function of sex, F (1, 82) = .337 p = .563, and there was no interaction between sex and treatment group, F(5,65) = .078, p = .995.
	There were no significant differences in center distance as a function of treatment group, F(5, 82) = .791, p = .559. There were no significant differences in center distance as a function of sex, F (1, 82) = .017, p = .896, and there was no interaction between sex and treatment group, F(5, 82) = .238, p = .945. 

Motor Coordination: Parallel Bar Success Ratio.

	There was no significant difference in Success Ratio as a function of treatment group, F(5, 97) = 1.040, p = .399. However, an interaction of Treatment by Sex was found, F(5, 97) = 3.810, p = .003. A post hoc was performed in order to find were the significant differences lay. Ethanol exposure significantly impaired motor performance in males, F(5, 45) = 3.395, p = .011, but not in females, F(5, 52) = 1.679, p = .156. This effect was attenuated by betaine administration in a dose-dependent manner.  Although 100 mg/kg/day betaine did not significantly improve performance, F(5,33) = 2.511, p>.05 (.369),  administration of 300 mg/kg/day of betaine, F(5,33) = 2.511, p<.05 (.030),  or 500 mg/kg/day, F(5,33) = 2.511, p<.05 did mitigate ethanol’s  adverse effects (see figure 4). 

Maximum Width.

	There was no significant difference in maximum width as a function of treatment group, F(5, 97) = 1.040, p = .399. However, an interaction of Treatment by Sex was found, F(5, 97) = 3.810, p = .003. A Tuckey post hoc was performed in order to find where the significant differences lay. Ethanol exposure significantly impaired motor performance in males, F(5, 45) = 3.395, p = .011, but not in females, F(5, 52) = 1.679, p = .156. This effect was attenuated by betaine administration in a dose-dependent manner.  Although 100 mg/kg/day betaine did not significantly improve performance, F(5,33) = 2.511, p>.05 (.369),  administration of 300 mg/kg/day of betaine, F(5,33) = 2.511, p<.05 (.030),  or 500 mg/kg/day, F(5,33) = 2.511, p<.05 did mitigate ethanol’s  adverse effects. 

Elevated Beam Task: Sensorimotor Coordination

	There were no significant differences in slip ratio as a function of treatment group (add statistics). This task was discontinued after a total of x litters. There was no ethanol effect in studies other than mine, so it was decided to be a useless task. 

Odor Recognition

	There were no significant differences in odor recognition memory as a function of treatment group. 

Object Recognition

	There were no significant differences in object recognition memory as a function of treatment group. 

Discussion

	Previous studies in our lab have shown that Choline supplementation during early development through to adolescence can attenuate the adverse effects of drinking alcohol during pregnancy and reduce brain abnormalities.  We predicted that choline affects the brain through the mechanism of methyl donation. If this is true, similar protective effects should be produced by the administration of Betaine since Betaine is a similar organic compound also involved in the Homocysteine Methionine cycle. Subjects were treated with alcohol during the 3rd trimester equivalent and given varying doses of betaine from PD 4-30. My preliminary data showed no significant results in the open-field activity testing, elevated beam task, or odor & object recognition. However my parallel bar results showed that males had significant motor-coordination impairments, and these deficits were attenuated in a dose dependent manner by betaine. Despite having a majority of insignificant results, I found apparent attenuative effects by Betaine in the ethanol-related behavioral alterations. 

Activity

	Although not significantly, Betaine helped to reduce the severity of overactivity associated with prenatal exposure, (see figures x and x). 

Fig C The x axis shows the treatment groups. The y axis shows the number of times subjects entered the center of the box. The higher the number, the more active the rats were, as is seen in the case of the ethanol-exposed rats given saline.

Fig D The x axis shows the treatment groups, and the y axis shows the total number of beam breaks made by the rat. The higher the number, the more activity the rat displayed. The figures above both show that the EtOH exposed group who was given no Betaine treatment seemed to have performed worse than all other groups. The Betaine 500 mg/kg dose looks as if its having a mitigative effect on the ethanol-related overactivity, although my preliminary data failed to provide significant results. What this most likely means is that in order for the apparent trend we see here to be statistically significant, more power is needed. Thus, as the number of subjects will increase with the progression of the study, both my standard error of measurement and my p value should decrease. Elevated Beam Task

	I decided to discontinue the running this task because my study, along with other studies, showed that the task wasn’t working. There was neither a main effect of ethanol, nor a clearly visible trend for Betaine. Thus, this task was considered obsolete and unusable. 

Parallel Bars

	Parallel bar results showed that compared to controls, subjects exposed to ethanol during their third-trimester human equivalent exhibited significant impairments in motor coordination. Betaine significantly reduced motor impairments in males who were exposed to alcohol prenatally, but not females. Ethanol exposed male subjects who were given 100mg/kg of Betaine showed no difference in success ratio compared to the EtOH-exposed + saline group.  Ethanol exposed male 

subjects who were treated with the 300mg/kg and 500mg/kg doses of Betaine performed better than the EtOH-exposed + saline group, but worse than the sham control group. Interestingly, however, Ethanol exposed female controls had a higher success ratio (35.85% vs. 16.75%), and a higher maximum width achieved (3.66 cm vs. 2.88 cm) than the males did. I believe these results can mean one of two different things. Perhaps physiological differences between male and female brains could be an important factor contributing to sex differences in motor coordination. For example, researchers have shown that there are sex differences in stress regulation, which are hormonally regulated through sub-cortical brain regions to cortical control of arousal (Goldstein, J.M., et. al., 2010). Their findings demonstrated that females have a natural hormonal capacity to regulate the stress response that is significantly different from the males. Perhaps the platform and rods which stood 63 cm above the wood chip bedding stressed the male rats to the extent that females were better at regulating the stress and thus were able to attain a higher maximum width and a better success ratio. The second possibility could be that male’s bigger size might also have an impact on the differences that we found between male and female parallel bar motor coordination. Moreover, having a greater mass to carry across the parallel bar apparatus might be one reason why it might’ve been harder for the males to traverse the bars. Odor and Object Recognition

	Results from both odor and object recognition showed no significant differences in odor and object memory. All of the treatments groups had no significant differences in the time they spent exploring novel object/odor 1 compared to novel object/odor 2. There were two factors I believe contributed to this memory task’s insignificant results. First, the procedure was not followed exactly as it had been recommended in Dr. Spinetta’s dissertation; where protocol for this task was taken from.  For some reason, the protocol in this lab did not think it was important to house one subject per cage.  Consequently, during the pre-habituation phase, the animals were not familiarized to four beads, but instead were sometimes exposed to even twelve beads simultaneously. The pre-habituation phase of the odor & object task is a critical phase. This is where the rats get familiar to either odors or objects. If you have a female rat for example, who was housed with two other rats, there might be a confound in the amount of time each rat spends familiarizing to the four beads. Perhaps some rats, only ever got to explore 3 of them, thus, acting as a confounding variable. Moreover, I believe that the fact that rats were sometimes familiarized to an inconsistent number of beads (not always the four beads stated in the protocol),  very likely influenced the results that I found. Secondly, since the animals were not ran during their light cycle, their energy and exploration levels were not at their optimal levels. In other words, perhaps  In sum, my preliminary data showed that the administration of betaine effectively reduced the severity of some behavioral alterations associated with developmental alcohol exposure, but not others. Our initial results have shown that betaine administration during and after alcohol exposure attenuates ethanol-related deficits in motor coordination. These results indicate that optimal levels of betaine can help reduce behavioral impairments associated with exposure to alcohol during pregnancy. However, at this point in our study, we have yet to determine the mechanisms of choline’s beneficial actions. Future studies should examine whether alcohol exposure influences betaine levels, and whether choline supplementation leads to increased betaine levels. If choline does not influence betaine levels, choline’s beneficial effects are most likely independent from that of Betaine’s. In conclusion, these findings have important implications for those individuals with FASD who can benefit from effective interventions. 

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