BIO 392 -WORM LAB (week 7)

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Central Michigan University *

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Mechanical Engineering

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Feb 20, 2024

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docx

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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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