BIO 392 -WORM LAB (week 7)

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Mechanical Engineering

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Feb 20, 2024

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Week 7 Earthworm Lab Report Goal: Create two experiments for inclusion in the earthworm action potential lab using mechanical stimulation and temperature as experimental variables. PART 1. SENSORY Objective: Create an experiment to determine whether action potentials can be evoked in medial and lateral giant fibers by determining sensitivity in different areas of Lumbricus terrestris (earthworm) with manual stimulation. Hypothesis: If an earthworm is manually stimulated with a wooden stick, there will be a larger medial and lateral amplitude recorded at the ends of the earthworm than in the center. Protocol: 1) Select the worm out of the dirt cup, and wash with warm water. Dry worm off, place the worm in carbonated water. Leave the worm in CO2 water for 4 minutes. Move the worm back into anesthesia if needed. 2) Place the black recording electrode 2 cm away from the end of the tail. Place the white electrode 4 cm away from the tail. Place the green electrode at 12 cm away from the tail. 3) Using a wooden stick, manually stimulate the end of the worm by lightly poking the tail, 2 cm away from the black electrode. Record the action potential amplitude in LabChart. Using a wooden stick, manually stimulate the middle of the worm at ½ the worm length. Record. Manually stimulate the head of the worm by lightly poking the tip opposite of the tail. Repeat step 3 twice more. Figure 1 : Experimental setup for mechanical stimulation of earth worm with wooden stick. Observations:

- The worm was moving slowly after 4 minutes in the carbonated water. The worm was placed in the carbonated water at 9:19 a.m. and removed at 9:23 a.m. After 6 minutes of being out of the carbonated water, the worm began to move again, so it was placed back in the carbonated water at 9:29 a.m. and removed at 9:32 a.m. When the worm was in the CO2 water, it expanded, and bubbles and crystals formed around it. When removed, the worm had a slimy filament on it. - The worm was 18 cm total, therefore the halfway stimulus point was 9 cm. - The green electrode did not show action potentials at 6 cm or 8 cm. Action potentials were found at 12 cm. - The worm was resubmerged at 9:58 a.m. and removed at 10:03 a.m. due to it moving around. Data: Distance From Tail; stimulation site (cm) Median Giant Fiber Amplitude ( µV) Lateral Giant fiber Amplitude ( µV) Round: 1 2 3 Average 1 2 3 Average 0cm 382.0 301.7 80.3 254.67 451.9 277.1 132.6 287.2 9 cm 31.0 31.2 57.7 39.9 51.5 51.2 44.8 49.17 18 cm 209.5 423.9 222.1 285.17 225.1 174.2 162.2 187.2 Findings and Interpretations: Our data consistently showed a significantly higher amplitude of action potentials when poking the ends of the worm, compared to the center with a stick. The average median giant fiber amplitude from the 3 tests at the tail end was 254.67 µV, and 285.17 µV at the head end. The average giant fiber amplitude reading for the middle of the worm was 39.9 µV. The average lateral giant fiber amplitude from the 3 tests at the tail end was 2872 µV, and 187.2 µV at the head end. The average lateral giant fiber amplitude from the middle of the worm was 49.17 µV. We interpret these findings as the head and tail of the worm being more sensitive than the middle to mechanical stimuli. This could mean that worms have a higher amount of mechanoreceptors in the tail and head regions. It was difficult to locate the exact median and lateral giant fiber amplitudes at 9 cm. This could be due to a lack of mechanoreceptor density or sensitivity in this area. However, there was a lot of variation within amplitudes for both the median and lateral giant fibers in the head (18cm) and tail (0cm). This variation could be explained by the worm moving and shifting the electrodes between recordings. Reflection:

The results aligned with our hypothesis. We assume the difference in sensitivity is due to the head and tail having a larger role in detecting environmental stimuli, compared to the middle of the worm. As the data suggests, there is a commonality in the amplitude readings for certain body areas, which we used to assess the amount of mechanical sensitivity in those areas. Worms have sensory neurons to detect stimulus in their environment. Their somatosensory mechanoreceptors that perceive mechanical stimuli are concentrated in areas of the body that help the organism move and detect. Worms have a larger portion of their nervous system at the head end, and may require sensory neurons to protect. PART 2. TEMPERATURE Objective: Create an experiment to determine whether chilling an earthworm will affect its ability to fire and conduct an action potential with electrical stimulation over time. Hypothesis: If earthworm temperature is decreased using ice, over time, it will take longer for action potentials of the median and lateral fibers to fire. As time on ice increases, latency will increase and conduction velocity will decrease with electrical stimuli implementation. Protocol: 1. Set up equipment and prepare the earthworm exactly as was done in last week’s experiment (Figure 2) a. Do not add extra dissecting pin at the head-end of the worm 2. Adjust pulse height on LabChart to 5V so that action potentials from the lateral and median giant axons are seen 3. Conduct baseline data when the worm is not on ice a. Using a ruler, record the distance between the white R1 and black stimulating cathode b. Record the time difference between the action potential peak from both the median and lateral giant axons and the stimulus artifact peak 4. Move the worm over the ice bath so its middle region is chilled and collect data (same as stated in step 3) every minute for 10 minutes a. This worked best because the middle region was able to be cooled while the ends of the worm were left to remain warmer and able to be pinned to the foam board. 5. Draw conclusions

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Figure 2. Experimental setup for earthworm preparation for electrical stimulation (without additional dissecting pin at the head-end) Data: Nerve Conduction Velocity Time on Ice (min) Nerve Distance (mm) Time (ms) Conduction Velocity (mm/ms) Baseline Median 76 4.7 16 Baseline Lateral 76 6.675 11 3 Median 105 16.475 6.37 3 Lateral 105 23.075 4.55 4 Median 105 18.5 5.68 4 Lateral 105 24.925 4.21 5 Median 105 15.9 6.60 5 Lateral 105 22.575 4.65 6 Median 105 15.275 6.87 6 Lateral 105 21.9 4.79 7 Median 105 16.15 6.50 7 Lateral 105 22.55 4.66 8 Median 105 16.9 6.21 8 Lateral 105 23.525 4.46 9 Median 105 16.875 6.22 9 Lateral 105 23.6 4.45

10 Median 105 17.55 5.98 10 Lateral 105 24.225 4.33 Observations: - Two worms had to be preapred during this experiment because the first worm used did not display any action potential despite our troubleshooting. The group concluded the worm may have been left sitting in the ethanol for too long. - For our second worm, the group was unable to get data as soon as it was placed on ice because initially it showed no action potentials to show. It took 3 minutes for me to adjust the cathodes and receiving electrodes so we could view action potentials of the median and lateral giant axons. - We initially wanted to record data for 5 minutes rather than 10 minutes, but since the latency values were inconsistent over time, we decided to retrieve more data. Findings: Conduction velocity decreased greatly from 0 to 3 minutes, was the lowest at 4 minutes, increased slightly from 5-6 minutes, then continued to slightly decrease. Data Interpretation/Reflection: The data followed a general trend of latency increasing and conduction velocity decreasing as the worm’s time on ice increased. The slight inconsistency of the data may be due to human error when determining latency. The markers and cursors may not have been at the absolute peaks of the stimulus artifact and action potential peaks. The retrieved data could have also revealed that worms simply reach their lowest action potentials after only 4 minutes on ice. After 4 minutes on ice, the latency of their action potentials fluctuate but follow a general upward trend. In general, this experiment concluded that time on ice affects the speed in which an action potential is fired by an earthworm. Tips for Future Students: - Ensure the worm is fully anesthetized before removing it from ethanol/soda water by poking the worm using a wooden probe. - Ensure that the electrodes are placed as medially on the worm as possible without puncturing the gut tube. POST-LAB QUESTIONS: 1. Did the amplitude of the action potential in the median giant axon vary with stimulus voltage? What is the physiological basis for your answer?

No, the amplitude of the action potential in the median giant axon did not vary with stimulus voltage. This is because the nerve fibers of the worm behave like action potentials from a single axon. Therefore, action potential amplitudes do not change with increasing or decreasing stimulus intensity/voltage. Earthworms essentially have one very large axon that produces action potentials when stimulated extracellularly. 2. Why did the latency differ between the median and lateral fibers? It takes longer for the action potential to peak in the lateral fibers because they are narrower than median fibers. This means it takes longer for the lateral fiber to conduct an action potential since the walls add resistance to ion flow. In this same sense, the medial fiber is larger than the lateral fibers so they are able to conduct an action potential quicker. 3. Why do you not see a second action potential from the median giant axon when the stimulus interval is very short? The nerve was in its absolute refractory period, so invoking an action potential was impossible no matter the strength of the stimulus due to voltage-gated sodium channels being inactivated. 4. Based on your data, what can you say about the directionality of the action potential? Based on our data, it can be determined that an action potential travels one direction at a time. This is because once a part of the membrane has depolarized, it then enters repolarization and the membrane enters its absolute refractory period. As depolarization spreads down the membrane, depolarization is blocked from returning to the area of the membrane in which it came from. The membrane cannot depolarize again because Na+ channels are inactivated and additional K+ channels have opened. 5. Develop two hypotheses explaining how low temperature affects fiber conduction. Explain as necessary. Are their data from earthworms/other invertebrates that support/refute your hypotheses? Cite at least one source. 1) If earthworm temperature is decreased using ice, over time, it will take longer for action potentials of the median and lateral fibers to fire. As time on ice increases, latency will increase and conduction velocity will decrease with electrical stimuli implementation. 2) If an earthworm moves from its optimal temperature of 10 degrees Celsius, then action potential latency will increase and conduction velocity will decrease as temperature moves away from optimal. Edwards, Clive A. and J. Lofty. “Biology

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