Roxana Bordbar

Melissa Marchand

November 23, 2011

The Effect of Caffeine on the Metabolic Rate and Wheel Running Speed of Mus musculus (House Mice).

Caffeine is a psychoactive stimulant that has been used throughout history by humans. In Mus musculus, it was expected that after ingestion of caffeine (4 grams/g body weight) the wheel running speed and the production of carbon dioxide, and therefore the metabolic rate would increase. Twenty minutes prior to data collection, each mouse was given a small piece of food and then after that time, was put on the wheel to retrieve data. The control trial was only given a small amount of food with .05 g of organic peanut butter. The experimental trial was also given a small amount of food with .05 g of peanut. However, this trial was also given .06 g of caffeine that was crushed and mixed into the peanut butter. After the twenty minutes, each mouse was then placed into the sealed container where carbon dioxide probes were inserted to calculate the carbon dioxide production. The data yielded a significant increase in the average speed of the mice after ingesting caffeine. The statistical analysis yielded a p value that determined a significant difference, p=.00062. Furthermore, there was a statistically significant difference between both the carbon dioxide produced and the metabolic rate between the control and experimental trials. The carbon dioxide produced yielded a p-value of .0001, and the metabolic rate also yielded a p-value of .0001, since carbon dioxide production was used to calculate the metabolic rate. The results of the study confirmed our hypothesis that caffeine increases wheel speed and the metabolic rate of Mus Musculus.

Introduction:

Caffeine is a bitter, white crystalline xanthine alkaloid and psychoactive stimulant (Fisone, 2004). In humans, caffeine acts as a stimulant to the central nervous system that temporarily provides energy, restores alertness, and increases metabolic rate (Poehlman, 1989). Caffeine works by having a mimicking effect of a hormone the body naturally produces. It is both water-soluble and lipid-soluble, so it readily crosses the blood–brain barrier that separates the bloodstream from the interior of the brain. Once in the brain, the primary mode of action is as a nonselective antagonist of adenosine receptors, which reduces the effects of the neurotransmitter adenosine. It binds to its receptors without activating them, making it a competitive inhibitor (Fisone, 2004). Furthermore, caffeine is the world's most widely consumed psychoactive substance, but, unlike many other drugs of its kind, it is legal for consumption. Beverages containing caffeine, such as coffee, tea, soft drinks, and energy drinks, are extremely popular all over the world and in North America, 90% of adults consume caffeine daily (Mcardle, 2010).

Because of its stimulating effects, caffeine is currently being studied by athletic boards because some argue that is has the potential to increase and amplify athletic ability (Monroe, 1998). These boards are often worried that it might give athletes an advantage over those who have not consumed caffeine prior to a competition. It has been a subject of debate as to whether athletes should be allowed to ingest caffeine prior to competitions. Thus, this study is aiming to determine whether this psychoactive substance really does have a distinguishable effect on one’s performance. Specifically, the investigators believe that using a caffeinated supplement will have an effect on the rate at which athletes perform in competitions.

Materials and Methods:

Ten house mice (Mus musculus) were purchased from Petco located in Mission Viejo, California, along with a cage, food, and a running wheel that measured 39.9 cm in circumference. The mice were all male and upon measurement found to have an average weight of 15.6 g. ALLMAX Nutrition caffeine tablets (75 mg) were also purchased from CVS pharmacy. Maranatha Organic Peanut Butter and Fiesta mouse food were used as well. Two Pasco Xplorer GLX carbon dioxide sensors, a barometer, and a thermometer were barrowed from the Saddleback College Department of Biology. A 750 mL enclosed, rectangular plastic container that was only slightly larger than the running wheel was used to provide an isolated environment for the mice. Two 3cm holes were drilled in the container where rubber stoppers for the probes were placed, and a 0.50 cm hole was drilled for the thermometer. The holes were drilled to allow the two carbon dioxide sensors and the thermometer to neatly fit helping prevent any air from seeping through the sides, creating a closed environment for carbon dioxide build-up. The volume remaining in the container was measured to be 316 mL after subtracting out the probes and the wheel. Prior to running experimental trails, an hour a day was spent training the mice to run on the wheel from November 4-6, 2011. To do this, positive reinforcement conditioning was conducted in the form of eatable treats. The experimental portion began on the 9th of November, 2011 at the house of Melissa Marchand, located in Rancho Santa Margarita, California. Located on site were two Pasco Xplorer GLX data loggers with the carbon dioxide probes, barometer, and thermometer. The two Pasco GLX probes were set up and calibrated in the plastic container prior to testing to verify all equipment was functioning properly. The ten mice were observed in the control condition on the first day of experimental data gathering. Prior to running on the wheel in the enclosed container, each mouse was given a single grain of their Fiesta mouse food with .05 g of Maranatha Organic Peanut Butter on top. Upon ingesting the grain of food, investigators timed twenty minutes for each mouse before experimentation began. After 20 minutes, each mouse was placed on the wheel in the container and subsequently began wheel activity. A timer was set for 5 minutes and measurement of carbon dioxide was recorded with the probes as an indicator of metabolic rate. Temperature and barometric pressure were taken prior to each trial as well. The numbers of total wheel rotations were counted in these 5 minutes in order to calculate running speed. The same procedure was repeated for each of the ten mice during day 1 of the experiment.

On the 10th of November, 2011, the same ten mice were put in the experimental condition. Prior to running on the wheel, each mouse was given a single grain of the Fiesta mouse food with .05 g of Maranatha Organic Peanut Butter. However, in this condition, there was .06 g of a crushed caffeine tablet mixed in with the peanut butter. This dose of caffeine was proportional to the average body weights of all of the mice (4 mg/g body weight) (Spriet, 1995). Upon ingesting the grain of food, investigators timed twenty minutes before experimentation began since caffeine’s optimal effects usually occur after twenty minutes (Liguori,1997). All other procedures were kept constant with the control condition.

The metabolic rate was calculated by taking the amount of carbon dioxide (ppm) produced over the five minute period. This was then converted to standard temperature and pressure using Henry’s Law. A paired, one-tailed t-test was used to determine statistical differences between the metabolic rate and running speed in the control trial and the experimental trial.

Results

The results obtained from the study are that of what was hypothesized. The mean speed for the experimental trial in which the ten mice ingested caffeine was significantly faster than that of the control trial, in which the mice received no caffeine. The average speed for the experimental trial was 2149 cm/ minute while the mice in the control trial had a mean speed of 1770 cm/ minute. A statistical analysis using a paired, one tailed t-test was performed to compare these means, which yielded a highly significant p value less than 0.05 (p= 0.00062).

Furthermore, there was a difference in production of carbon dioxide between the two trials. Specifically, there was slightly more carbon dioxide produced from the experimental run than the control run. The average mean carbon dioxide produced for the caffeinated run was 3,476.5 ppm (parts per million) while the control run had an average production of 3,650.3 ppm. A paired, one tail t- test was also performed to compare the two means, and there was found to be a significant difference between the two means (p=.0001).

The carbon dioxide production was used to calculate the actual metabolic rate produced by each trial. The metabolic rate was calculated by using the barometric pressure, the temperature of the enclosed container in Kelvin, and the carbon dioxide produced per minute per ml. This was all compared to the standard pressure and temperature to then find the metabolic rate. There was found to be a statistically significant differences between the means of the experimental trial and control trial for metabolic rate as well, with a p value of less than .05 (p=.0001).

Figure 1. Comparison of average wheel speeds of (Mus musculus) with and without caffeine ingestion. P value less than 0.5 yields significant difference between the two groups (p=0.00062).

Figure 2. Comparison of average carbon dioxide production means in parts per million before and after caffeine ingestion. P value less than 0.5 yields significant difference (p=.0001)

Figure 3. Metabolic rates compared between both trials that received caffeine and that did not receive caffeine. Means were compared for statistical analysis. P values less than 0.5 yield a significant difference (p=.0001). Both means and standard mean errors are shown.

Discussion

The increase in speed and metabolic rate of Mus musculus was influenced by their ingestion of caffeine prior to wheel activity. The increase in their speed while running on the mouse wheel, as well as the increase in their carbon dioxide production, indicated the physiological effects of caffeine. The production of carbon dioxide (ppm) was used in the calculations for metabolic rate (figure 2), and the wheel revolutions were used in calculating the running speed (figure 1). Speed was significantly greater in the experimental trial, where caffeine was ingested, as opposed to the control trial which received no caffeine. Metabolic rate was also significantly greater in the experimental trial, as opposed to the control trial, which confirmed the hypothesis tested.

Previous studies have shown similar results, indicating that caffeine enhances neurological alertness, endurance in exercise, and increases in metabolism (Spriet, 1995). The study conducted, aimed to not only determine if caffeine increased metabolic rate, but also whether it increased wheel running speed, specifically, rather than endurance. Studies have also discussed the mechanisms by which caffeine works to enhance various aspects of the body’s physiology. Specifically, caffeine functions by acting as a competitive inhibitor of the molecule adenosine (Nehlig, 1992). By binding the A-1 and A-2 receptors, the caffeine works to block the binding of the adenosine to its receptors, which disallows the effect of adenosine on the body (Basheer, 2004). Because adenosine is inhibitory neurotransmitter that suppresses neural activity, promotes sleep, and suppresses arousal, blocking its effects increases arousal and activity (Peters, 1967). Thus, the caffeine in the nervous system lowers the perception of effort by lowering the neuron activation threshold, allowing the enhancement of muscle activity.

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