Animal Behavior Research Project:

The Effect of Bacopa monniera on Spiny Mice

Brianna Clark

BI441/PSY441

Spring 2004

 

Abstract

 

In this experiment I tested to see whether or not the herb, Bacopa monniera, would affect learning behavior and memory in spiny mice (Acomys sp.). My main concern was to observe if there was a difference in the rate of learning/memory between two groups of spiny mice, one control group given 15 mL of regular, fresh tap water as part of their daily diet and one experimental group with 0.24 mg of powdered Bacopa monniera extract rationed in with the daily 15 mL fresh tap water of their diet. I measured the progression of learning/memory enhancement by administering nine tests in which I recorded the amount of time that it took each of the eight individual spiny mice to run and complete the same complex maze. After administering numerous trials over the designated period of time and a t-test that generated a p-value equal to 0.01, it became evident to me that the mice in the Bacopa monniera (experimental) group learned to finish the complex maze faster. Thus, the addition of Bacopa monniera to a spiny mouse’s diet genuinely did effect and enhance memory.

 

Keywords: Bacopa monniera, Acomys sp., spiny mice, learning behavior, memory

 

Introduction

Since the sixth century, Bacopa monniera has traditionally been utilized to support mental clarity and alertness. Mainly grown and used in India, this “Brahmi” herb is claimed to not only heighten short and long-term memory, but also support the physiological processes involved in relaxation. Furthermore, it is considered the major rejuvenation herb for nerve and brain cells. Therefore Bacopa monniera has been extremely important in Ayurvedic therapies, especially for the treatment of epilepsy, insomnia and cognitive disorders associated with aging (Russo et al. 2003). Recent studies have demonstrated that extracts of Bacopa monniera reduces the memory-dysfunction in rat models of Alzheimer's disease, but the molecular mechanisms of this action are yet to be determined (Russo et al. 2003).

However in relation, research has found two chemicals in Bacopa monniera (bacosides A and B) that are responsible for improved transmission of impulses between nerve cells in one’s brain. These bacosides are vital in the progression of learning and remembering new things because they are capable of regenerating synapses and repairing damaged neurons (Ellis 2002). Likewise, learning and memory are based on modifications of synaptic strength among neurons that are simultaneously active (Tang et al. 1999). Overall, recent studies have reported that extracts of Bacopa monniera have had cognitive enhancing effects in animals (Stough et al. 2001).

Due to these investigations, along with Tang et al. (1999) stating that mice exhibit superior ability in learning and memory in various behavioral tasks, I designed this experiment in order to test whether or not the Bacopa monniera herb would affect the learning behavior and memory in spiny mice (Acomys sp.).

If Bacopa monniera does have an affect on learning behavior and memory, I expect to see a difference in the rate of learning/memory between the two groups of spiny mice. Over time, the experimental group of spiny mice whose diet contains Bacopa monniera should tend to learn to complete the complex maze at a quicker pace than the control group of spiny mice.

Materials

* 8 Spiny Mice (Acomys sp.)                                   * Stopwatch           

* 4 Plastic Cages with Metal Wire Covers            * Labeling Tags/Tape

* 4 500-mL Plastic Water Bottles                          * Permanent Sharpie Marker

* 3 1000-mL Glass Flasks                                      * Pine Shavings/Bedding

* 2 500-mL Glass Beakers                                     * Fresh Tap Water

* 1 1000-mL Glass Beaker                                     * Complex Maze with Plastic Cover

* 1 100-mL Glass Graduated Cylinder                  * Square/Block Chow

* Analytic Scale                                                        * Peanut Butter

* Metric Mass Scale                                                * Oatmeal

* Cleaning Supplies                                                 * Aluminum Foil

* Bacopa monniera – Pure Extract Capsules      * Meter Stick

* Stirring Plate with Magnetic Stirring Rod           * Refrigerator

*Microsoft Excel (to make tables, a graph and implement statistical tests)

                 

Methods

I started this experiment out by writing up a research proposal and then shortly thereafter got it approved to be both a practical and ethical experimental study by the Assistant Professor of the Biology Department at Alverno College, Rebecca Burton, Ph.D. Once this was accomplished, I began my project on Monday March 15, 2004. I gathered four plastic cages with metal wire lids that already housed a total of eight spiny mice. Cage 1 contained two “supposed” female spiny mice (Figure 1), cage 2 housed two male spiny mice (Figure 1), cage 3 had three spiny mice females (Figure 2) and lastly a solitary male spiny mouse occupied cage 4 (Figure 2). I then labeled the four cages by attaching tags with the above information, assigned each mouse a number (1-8) and wrote which of the two groups that each cage was going to be in. The experimental Bacopa monniera group became cages 1 and 2, while the control, NO Bacopa monniera group then contained cages 3 and 4. Following this extensive labeling process, I supplied each of the four cages with 70 g of square/block chow and 100 mL of fresh tap water to last until the next day (Tuesday March 16, 2004); this was when I was going to start the difference in liquid diet between the two groups. The last task of Monday was to mass each mouse and record it in my notebook. Then on Tuesday March 16, 2004, I began with the mouse mass data that I had ended with on the day before. I took these previously obtained eight masses, found an average (54.5 g) and then calculated the precise dosage of powdered Bacopa monniera extract that an “average” spiny mouse should consume on a daily basis. This was based on “average” mouse weight in comparison to “average” human weight (68 kg)/suggested dosage of powdered Bacopa monniera extract  (300 mg per day). Thus, a proportion was determined. After a few calculations, I concluded that every spiny mouse in the experimental group needed 0.24 mg of powdered Bacopa monniera extract per 15 mL of fresh tap water daily. From here, I decided that it was important for me to make stock flasks of the powdered Bacopa monniera extract/fresh tap water solution. I wanted to keep all cage set-ups equal no matter how many mice were in each one. So to remain consistent, I assumed that each of the cages contained three mice. That way no mouse would ever have the chance of going hungry or thirsty during a week span, nor did one cage get more or less than the rest. I did some more calculations and determined that each of the four mice in cages 1 and 2 individually needed 1.68 mg of powdered Bacopa monniera extract added to 105 mL of fresh tap water per week. That meant that cages 1 and 2 each needed a 500 mL water bottle full of a 5.04 mg powdered Bacopa monniera extract/315 mL fresh tap water solution. On the other hand, cages 3 and 4 required just the 315 mL fresh tap water per each bottle every week. From here, I used three 1000 mL flasks and made the powdered Bacopa monniera extract/fresh tap water solution in each according to my figures that stated that each flask should contain 990 mL of fresh tap water and 15.84 mg of powdered Bacopa monniera extract. To better mix the powdered extract and the fresh tap water, I placed each flask onto a large stir plate and allowed them each to mix for a total run time of five minutes (I continued to do this throughout the course of this experiment. Whenever I needed to measure out this Bacopa monniera extract/fresh tap water solution for cages 1 and 2, I would repeat this step of mixing on a stir plate). After the solutions were mixed thoroughly, I emptied out the regular fresh tap water from all four of the cage water bottles and then filled each according to what group the cage belonged to (cages 1 and 2 each got 315 mL of the powdered Bacopa monniera extract/fresh tap water solution, while cages 3 and 4 got 315 mL of plain tap water). Also, I labeled each water bottle with what specifically was in each one. To eliminate growth and contamination, I then covered the tops of the three powdered Bacopa monniera extract/fresh tap water solution flasks with aluminum foil and stored them in the Cell Biology/Molecular Biology refrigerator. In addition to making stock flasks and filling each of the four water bottles with their designated solution, I massed out 147 g of the square/block chow for each cage. Before I left for the day, I covered the bottom of four clean cages and then transferred the eight mice from the four previous cages into my new, fresh set-ups (Figures 1 and 2). Lastly, I made sure to put the metal wire lids with the label tags on the correct new cages. When I came in on Wednesday March 17, 2004, I started off by making 72 – 1 g peanut butter and oatmeal treats. Once this activity was completed, I began my first maze trial. I gathered the complex maze set-up (Figure 3), the clear plastic cover (Figure 3), a stopwatch, a 500 mL beaker and eight peanut butter and oatmeal treats (which were positioned at the end of the complex maze, in the upper right hand corner). One at a time, I placed mice into the complex maze and allowed him or her to run it at their own pace. Timing started as soon as the mouse was placed in the upper left hand corner of the complex maze and the clear plastic cover was securely on top. I considered them to have completed the maze and stopped the stopwatch when they would turn the corner in the upper right hand corner of the complex maze and touch the peanut butter oatmeal treat. Between each mouse, I made sure to clean up any urine, droppings or pine shavings/bedding with paper toweling. Each mouse ran the maze only once. I made sure to record the times obtained in my laboratory notebook. After I cleaned up and before I left, I did a quick check to make sure there was enough food and water to last them overnight.  When I returned on Thursday March 18, 2004 and Friday March 19, 2004, I repeated the same steps regarding the maze trial as I had performed on Wednesday and remembered to do a rapid check of their food/water supply before leaving. I did not come in on Saturdays or Sundays during this research study since I felt that there was enough food and water to get them through the weekend. However when I did finally return on Monday March 22, 2004 to verify that the mice had enough food and water to last them until Tuesday, cage 1 had two adults and three new baby mice. Due to this fact, I removed mouse 2 (assumed to be the father) from this cage and placed him into a new, clean one in order to keep him in the experiment. I transferred the metal wire cover with the calculated food, water, and the labeling tag. Additionally, I re-marked it to indicate that this was still cage 1 and belonged in the Bacopa monniera (experimental) group but instead contained one male spiny mouse that was going to have to live alone for the rest of my project. I gathered a new lid and made a new tag for the “old” cage, which I labeled to show that this set-up was no longer part of my experiment and that there was a mother with three babies in it. I did this in order for her to raise her babies in peace. I was now down to three mice in my experimental group (one less than the control group). Once I informed my instructor of the situation, I left for the day. I returned on Tuesday March 23, 2004 to clean the four cages, measure how much food/water the mice were consuming a week (to make sure they were getting the recommended dosages), give each cage 147 g of fresh square/block chow and fill the 500 mL bottles of water with 315 mL of the assigned water solution. Then on Wednesday (March 24), Thursday (March 25), and Friday (March 26) I came in to not only check the food/water levels, but to run one maze trial (per day) just as I had done during the previous week. Once again, I did not come in on the weekend; instead I came back on Monday (March 29) to check the food/water consumption. On Tuesday (March 30), I repeated the same steps that I had on the Tuesday prior (cleaned cages, measured the actual amount of food/water used and gave each cage fresh food/water according to plans). Wednesday (March 31), Thursday (April 1), and Friday (April 2) were the last three days of maze trials. I simply followed the same procedure as before. I did return once again on Tuesday April 6 to do the last measurements on food/water consumption and clean up all materials that I used in order to end my experiment and return everything back to the college. Overall, I ran this experiment for a total of 3 weeks. I made sure to record all of my data and observations in my laboratory notebook. Lastly, I used Microsoft Excel to make a few charts, graph my results and generate a p-value by means of a type 3 t-test. For this statistical test, I assumed that array 1 equaled the Bacopa monniera (experimental) group, array 2 equaled the NO Bacopa monniera (control) group, tails equaled 1 and type was 3.

 

Figure 1: Bacopa monniera (experimental) group set-up for cages 1 and 2.

 

 

 

 

 

Figure 2: NO Bacopa monniera (control) group set-up for cages 3 and 4.

Figure 3: Complex maze set-up. Box dimensions were 66.1 cm long x 51 cm wide x 25.8 cm high. The top left vertical divider was 12.5 cm from the left vertical wall and 24 cm in length. The bottom left vertical divider was 23 cm from the left vertical wall and 24 cm in length. The top middle vertical divider was 38 cm from the left vertical wall and 28 cm in length. The bottom right vertical divider was 39.5 cm away from the left vertical wall and 21 cm in length. The top right vertical divider was 53.5 cm away from the left vertical wall and 10 cm in length. The top right horizontal divider was 10 cm away from the top horizontal wall and 8 cm in length. All dividers had a thickness of 3 mm and a height of 7cm. Dimensions of the clear plastic cover that was placed on top of the complex maze were 91.5 cm x 91.5 cm.

 

 

Results/Data

In general, the mice in the Bacopa monniera (experimental) group learned to run and complete the maze at a more rapid rate than the NO Bacopa monniera (control) group. I charted (Tables 1 and 2) and then graphed the average times to complete the complex maze for the two groups over a period of nine trials (Figure 4) and noticed the variation amongst the two lines that were created. Next I ran a type 3 t-test and gained a p-value of 0.01. Since this is less than 0.05, I concluded that the results that were obtained were significant. Between the two groups tested, there was an evident difference in the time (averages) that it took the spiny mice to finish the complex maze.  

 

 

 

Table 1: Individual times in seconds for the spiny mice to complete the complex maze set-up for a series of nine trials. Mice 1 through 4 were in the Bacopa monniera (experimental) group, while mice 5 through 8 were in the NO Bacopa monniera (control) group.                                          

	Bacopa monniera (experimental)				NO Bacopa monniera (control)
Trial Number	Mouse 1	Mouse 2	Mouse 3	Mouse 4	Mouse 5	Mouse 6	Mouse 7	Mouse 8
1	42.67	20.34	38.03	53.62	486.78	128.07	75.09	33.18
2	38.23	50.28	16.5	27.37	177.28	208.75	44.63	6.87
3	34.63	28.37	17.93	9.65	30.21	151.75	34.03	65.35
4	n/a	25.66	15.13	41	125.87	61.85	270.81	31.28
5	n/a	24.61	5.32	8.03	32.75	106.07	52.03	45.84
6	n/a	7.22	6.35	6.22	15.41	24.59	17.88	30.27
7	n/a	8.1	6.84	10.09	39.35	23.19	17.62	53.1
8	n/a	55.81	19.53	7.84	42.69	41.84	26.78	37.72
9	n/a	45.09	9.03	2.66	34.97	39.5	17.28	15.59

 

 

 

 

Table 2: Average times in seconds of the two groups at each trial. Averages of the B. monniera group (experimental) do not contain any data for mouse 1 since she had babies and was removed from the research study.

Trial Number	Average of B. monniera Group (experimental)	Average of NO B. monniera Group (control)
1	38.67	180.78
2	33.10	109.38
3	22.65	70.34
4	27.26	122.45
5	12.65	59.17
6	6.60	22.04
7	8.34	33.32
8	27.73	37.26
9	18.93	26.84

Figure 4: Effect of Bacopa monniera on learning behavior and memory of spiny mice. Experimental (green line) = Bacopa monniera group, control (blue line)= NO Bacopa monniera group. Overall, there is a significant difference between the two lines. The experimental group line was notably lower than the control group line.

 

Discussion

While trying to establish and begin this experiment, I struggled with certain set up aspects. First of all, I had a hard time finding just the right supplies for what I wanted to test. Bacopa monniera was a hard herb to locate and purchase in Milwaukee. After some extensive shopping I ultimately found it, but then ran into dilemmas dealing with mouse sample size, creating the solution of Bacopa monniera, choosing a maze set up, and preparing treats for the mice. Overall, the design and implementation took up a few days of consecutive planning. Nevertheless, once I finally got started and was able to independently carry out the study I gained valuable and significant data demonstrating that the mice in the Bacopa monniera (experimental) group eventually learned to complete the complex maze at a faster rate than the control group. Thus, the addition of Bacopa monniera to a spiny mouse’s diet certainly did effect and enhance memory.

Fortunately for me this does relate to known findings such as, the notion that mice exhibit superior ability in learning/memory in various behavioral tasks (Tang et al. 1999) and more importantly that extracts of Bacopa monniera have memory enhancing effects in animals (Stough et al. 2001). For my specific experiment, mice in the Bacopa monniera (experimental) group tended to run and complete the complex maze faster, which leads one to believe that the herb affected their learning behavior and consequently their memory. Overall, I feel that the evidence and data collected genuinely support my hypothesis. The herb, Bacopa monniera essentially improves memory in animals.

If I were to repeat this experiment again in the future, there are a few aspects that I would want to alter before beginning. Once again, sample size would be one of them. I only used a total of eight spiny mice due to time constraints, amount of habitat/storage space available and use of mice by other groups. It did not help that one mouse from the experimental group had babies early on and had to be removed from the study. Thus, lowering my sample size to seven. In my opinion, this is a really small sample size and I truly need more spiny mice to gain a better representation, more support, as well as, a greater understanding of their learning behavior and capacity for memory enhancement.

Another portion that I would change would be to alter the set-up of the plastic cages. As I mentioned earlier, mouse 1 was unknowingly pregnant and delivered three babies within the first week of the study (March 22, 2004). At this point in time, I am uncertain if mouse 1 happened to get pregnant while still at the pet store or if the cages were labeled wrong (one male and one female, instead of two females) and she became pregnant in the animal behavior room of Alverno College. Due to this incident, I would prefer to place the spiny mice into their own separate cages. In addition, I would care to have equal number of males and females in each of the two groups, in order to see if there is a difference between the sexes.

It would be interesting but more importantly, beneficial if I would increase the duration of the research project. I chose three weeks because of the amount of time that was left after planning and establishing the work at hand. Honestly, I felt that this was not an adequate timeframe to truly test to see whether the Bacopa monniera herb affects memory. Instead, I would prefer an interval of study that lasts six to eight weeks in possible future studies.

In relation to this, I would rather be able to work with a partner. I began this experimental design with another individual. Unfortunately for me, she decided to drop the course. I was fascinated by the topic of improving memory through herbal supplements, thus I did not dismiss my proposal and carried on with the research by myself. Honestly it was a lot of work, not to mention, stress. It would have been nice to have someone there with me to help with the covering of the complex maze and timing each of the test subjects. In general, I believe this would have made the experiment move at a faster yet more organized rate.      

The final facet that I would modify deals with establishing a true “control” at the beginning of the study. I would want to feed all of the spiny mice fresh tap water for a period of one week and administer three complex maze trials per each mouse during this block of time. I would do this with the intention of determining if there was an actual discrepancy between the two groups in the first place.

 

Literature Cited

 

Ellis, L.  2002.  Improve memory with Bacopa.  Natural Health, 32: 29. Retrieved February 3, 2004 from the EBSCOhost database.

 

Russo, A.; Borrelli, F.; Campisi, A.; Acquaviva, R.; Raciti, G.; and A. Vanella.  2003.   Nitric oxide – related toxicity in cultured astrocytes: effect of Bacopa monniera. Life Sciences, 73: 1517-1526. Retrieved May 4, 2004 from the BasicBIOSIS  database.

 

Stough, C.; Lloyd, J.; Clarke, J.; Downey, L.; Hutchison, C.; Rodgers, T.; and P. Nathan.  2001.  The chronic effects of an extract of Bacopa monniera (Brahmi) on   cognitive function in healthy human subjects.  Psychopharmacology, 156: 481- 484. Retrieved February 3, 2004 from the InfoTrac database.

 

Tang, Y.; Shimizu, E.; Dube, G.; Rampon, C.; Kerchner, G.; Zhuo, M.; Liu, G.; and J. Tsien.  1999.  Genetic enhancement of learning and memory in mice.  Nature, 401: 63-70. Retrieved March 22, 2004 from the EBSCOhost database.