Abstract

The purpose of this lab was to study the behavior of action potentials in earthworms. Earthworms are ideal for the study of action potential conduction velocity because of their simple structured, easily measured bodies. Earthworms have two giant fiber nerve systems, lateral and medial, in which they conduct velocities that are easily measured through electrode manipulation. To indicate the action potential, axon myelination and diameter size are important factors. According to some studies, a larger axon diameter has resulted in a faster action potential in the axon. Most importantly, tripling the myelin thickness increases the conduction velocity by 3 times, whereas tripling the axon diameter resulted in an increase to the conduction velocity by square root of 3, or 1.7 times (CCNY BIO. 85). The hypothesis made for this study was that the medial giant nerve, MGN, would have a more rapid conduction velocity speed than the lateral giant nerve, LGN, due to a thicker myelin. In this experiment, using a Spike Recorder, the conduction velocity of earthworms were measured from both anterior and posterior ends. As hypothesized, the mean conduction velocity was significantly higher in the MGN than in the LGN.

Introduction

 In this lab, the behavior of action potentials were studied in earthworms. Action potentials are the electrical currents that are made by our nerves when we receive a stimulus, allowing us to feel things and make movements. The medial and lateral giant nerves both transmit various sensory information from various parts of the worm’s body.

 In order for the occurence of an action potential, a threshold value must be reached by the stimulus. Action potentials happen when the sodium channels are activated on a the membrane  causing a high permeability to sodium ions, which causes the membrane to depolarize. When potassium channels open and sodium channels close, the membrane begins to repolarize, allowing it to return to its resting potential. (Russell et al. 2016). During this process, only one stimulus can create an action potential at a time and a second one cannot be generated with any stimulus; this is known as the refractory period.

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 During this lab, the main focus was to view the action potential of the worms and to calculate the conduction velocities of both the Medial and Lateral axons. Although action potentials usually only travel in one direction, since we are manually stimulating the worms, the action potential can be measured in both directions with modifications to the electrodes. Keeping into mind that the LGN transmits sensory information from the posterior end to the anterior end, while the MGN transmits sensory information form the anterior end to the posterior end, allowing for easily measured muscle contractions. The class hypothesized that the conduction velocity of MGN would conduct a faster action potential than the LGN, due to the MGN having a larger axon diameter. Typically, as axon increases its diameter, its myelin thickness also increases. Perhaps the MGN has a thicker myelin sheath as well (Backyard Brains. 2019).

Materials and Methods

         In this experiment, the materials that will be the Lumbricus terristrius, known as an earthworm. We will also be using 10% ethanol solution, the Faraday cage and 2 channel SpikerBox, a piece of balsa wood or thick cork, USB cable, Laptop with Spike Recorder software, ruler, tape and marker.

In all experiments in this report, earthworms were provided to conduct readings on. The healthy earthworms were placed into 10% ethanol solutions for 2-3 minutes each so that they could be anesthetized. They were originally supposed to be left in this ethanol solution for 7-10 minutes; however, they were not left in there for that long because too much anesthesia would cause nerves to not fire, and too little anesthesia would cause movement during the experiment, resulting in muscle electrical activity, which will mess with the small neural electrical signals that are meant to be obtained (CCNY Bio Lab Manual. 73). Shortly after the earthworm stopped moving, they were placed into a beaker of distilled tap water for a few seconds just to rinse their bodies off. The earthworm was then placed onto a piece of balsa wood, which was covered in saran wrap for preservation, dorsal side up and was kept moistened throughout the experiment by wetting a piece of tissue and delicately touching their skin, making sure to avoid the upcoming electrodes. The balsawood was then placed into a Faraday cage, which was easily accessible from anterior and posterior ends. After that, with the two-channel SpikerBox, the 3 electrodes were placed, slightly off the centerline (to avoid piercing intestines or ventral nerve cord), into the anterior end of the worm. The red and white electrode were 0.5 inches apart and the white and black were 1.5 inches apart. The electrodes were plugged into the Neuron SpikerBox Pro and the USB cable into a PC. Open the  Spiker Recorder software and click on the USB symbol to pair with the Neuron Spiker Box. Next, press the record button and using a plastic or wooden probe, gently tap the anterior end of the worm. Once several spikes were obtained, they were recorded. This procedure we then repeated when trying to obtain recording from the posterior end. Once completed, the worms were returned back into their soil. They make look really tired and worn out but the worms are able to handle a lot and recover well from the experiments.

Results

Figure 1. This chart represents the average measurements of the lateral and medial giant fiber conduction velocities in the earthworm; along with the standard deviation.

In Figure 1, the data represents the means and standard deviations of the medial and lateral giant nerve fibers. The mean conduction velocity of the MGN, 14.5 m/s, with a standard deviation of 3.2, was significantly higher than that of the LGN, 8.52 m/s, whose standard deviation was 1.98.

T-Calculated

5.71

T-Critical for 95% Confidence Level

1.71

Degrees of Freedom

24

Confidence Level

97.5%< x <99%

Table 1. This chart represents the statistical analysis between the average of both groups, MGN and LGN.

Table 1 represents the statistical analysis that was conducted using the mean, standard deviation and sample sizing from both MGN and LGN. According to the table, T-Calc had a value of 5.71. There were 2 sample sizes of 13, leading to a value of 24 degrees of freedom. With a 5% confidence level/ p-value range, it is shown that the t-critical value, 1.71 was less than the t-calc value. This means that our data is significant, leaving us with the confidence level of being >97.5% but <99%.

Discussion

In conclusion, it was found that the MGN, anterior end, conduction velocity was indeed faster than the LGN, the posterior end. Our hypothesis that the MGN would transport a faster action potential than the LGN has been proven true. This hypothesis is supported by both results provided, along with background information, since the data is statistically significant, which is shown by the error bars in Figure 1. Also, t-calc is greater than t-critical with above a 95% confidence level.

Possible Errors

 In every experiment, there are always a vast amount of opportunities to cause errors, whether human or experimental. Many errors could have derived from using the Spike Recorder. A person conducting this experiment might have a heavy hand, so their force could have an affect on the action potential because of a greater stimulus to the worm unlike someone with a more delicate touch. Another possible error could be with the measurements of the distance and the placement of the electrodes. Maybe a group could have been reading the data backwards, so while doing anterior readings, their electrodes were placed in the order for reading posterior spikes. Some other ways to go about this experiment would be to try different organisms to experiment on.

Bibliography

Shannon, K. M., Gage, G. J., Jankovic, A., Wilson, W. J., & Marzullo, T. C. (2014). Portable conduction velocity experiments using earthworms for the college and high school               neuroscience teaching laboratory. Advances in physiology education, 38(1), 62–70.                             doi:10.1152/advan.00088.2013

Course Supplement for Biological Foundation 1, Biology 10200, Department of Biology, City​College of New York. (2019).

Brains, B. (n.d.). Experiment: Comparing Speeds of Two Nerve Fiber Sizes. Retrieved April 12, 2019, from https://backyardbrains.com/experiments/comparingnervespeed.

Russell, P. J., Hertz, P.E., & McMillan B. (2016). Biology: The Dynamic Science (4th ed.). Boston, MA: Cengage Learning.