UW Biology of Addiction Lab 5: Worms on Drugs UW Biology of Addiction

Learning Targets Hypothesis: I can predict whether my worm was given a stimulant, depressant or the placebo by examining pulse rate changes in the worms Success Criteria Observe the effects of drugs on the physiological function of living organisms Perform double-blind experiments Obtain, refine and analyze quantitative data Investigate blood pressure and pulse as peripheral measures of drug action

Materials Lumbriculus variegatus (California Blackworms) Stereoscope Wax slide with grooves Disposable pipettes to transfer worms Disposable pipette to dose worms Mystery solutions: A1, A2, B1, B2, C1, C2, C3 Timer Pond water Drugging chambers Detox Chamber Blood Pressure Monitor Caffeinated soda De-Caffeinated soda Data tables ready

Model I: PreLab http://www.eeob.iastate.edu/faculty/Drew esC/htdocs/INT-ANIMA-dbv-vel.htm Q1: What happens to this blood vessel? What physiological purpose does it serve (hint: this worm has no heart)? A1:

Model II Q2: What can you conclude about the impact of this drug on systolic blood pressure? Be thorough and consider what the * means. A2: Q3: If you didn’t already consider this, what is the placebo and why is it used? A3: Q4:Is this drug a stimulant or depressant? How can you tell? A4: Now analyze the following graph…

Model II: Pre-Lab http://www.eeob.iastate.edu/fa culty/DrewesC/htdocs/INT- ANIMA-LvDBV-mid.htm Q6:How many events occur in a minute? Come up with 3-4 things that might change this rate A6: For Q6, “events” refers to the organisms pulse which looks like an eye winking To answer Q6, we will observe pulses occurring in 10 seconds, 3xs and take the average We will measure beats per minute for each worm separately, BEFORE and AFTER drug treatment Q7: Why do a before and after test on each worm? A7:

Background: Careful with the live subjects Read over lab instructions regarding the worms. They are very sensitive to stimulation of all types Steady contact with worm, pipette and slide Darkness turn off the stereoscope when not in use and keep the light as dim as you can.

Background: Double Blind Experiments Neither subjects nor researchers know who’s in the control group and who’s in the experimental group. Experimental Group= given MV, but doesn’t know it Control Group= given placebo, but doesn’t‘ know it Researcher doesn’t know which subjects are in the experimental group or in the control group

Background: Double Blind Experiments Why do we use a double blind experiment? Avoid placebo effect, to control for subject’s and researcher’s expectations Placebo = A type of control In medicine, substance that’s pharmacologically inactive but could result in therapeutic effect soley based on power of suggestion Are 2 experiments in this week’s lab double-blind or not, why? you won’t know which drug you are giving to your worm (researcher blind), neither does your worm (subject blind) you won’t know which kind of cola you’re drinking (subject blind), neither does your lab partner (researcher blind)

Part I (Procedure) Become comfortable adjusting and focusing the stereoscope. Keep the light as dim as possible while working with the worm. Obtain 1 worm from worm tank: Using a wax covered slide and the worm transfer pipette, gently suck up ONE worm into the pipette. Align the pipette tip with the slot on the slide and release the worm into the slot. Let excess water roll off. Use the tip of the pipette to gently nudge the worm into position as needed. Now you are ready to get baseline pulse rates.

Part I (Procedure) To get the worms baseline pulse rate: Calculate beats/min by counting how many “pulses” you observe in 30 seconds and x 2. Take several measurements (at least 3) and average them. Do this for each worm separately. Be sure to record on the data table.

Part I (Procedure) Drug your worm in the following way: Take up a small squirt of your designated drug. Use the labeled pipette for the drug you were assigned to use. Hold the slide/worm over the drugging chamber at an angle and squirt the solution onto the worm, washing it down into the drugging chamber.

Part I (Procedure) Wait 15 minutes for the drug to take effect on the worm and recount the worm’s pulse post drugging. Do this 3 times and average, recording these pulse rates on the data table. When you are done obtaining data on this worm, flush it down into the detox beaker with pond water. Repeat this procedure for your second worm and assigned drug. Detox Beaker

Data Tables

Per. 2 Drug Assignments & Results Group A1 % Change A2 B1 B2 C1 C2 C3 1-Rashell, Alexis & Corey 78% 9% 2-Megan, McLaine, Maddie 85% 69% 3-Maddy, Lauren & Andrew 34% 12% 4-Miguel, David & Vincent 133% 42.8% 5-Nate & Austin 36% 56% 6-Amanda & Natalie 0% 58% Average %Change 106% 61% 17% 43% 33% 64%

Data Tables Period 2 Code A1 A2 B1 B2 C1 C2 C3 What drug was it? 2mM Caffeine 4mM Caffeine 0.005mM Nicotine 0.05 mM Nicotine Pond Water 1:40 dilution Ethanol 1:20 dilution Ethanol Pulse Change 106% 61% 17% 43% 33% 64% 12%

Per. 3 Drug Assignments & Results Group A1 % Change A2 B1 B2 C1 C2 C3 1- Indi, Bri & Shelby 81% 6.3% 2- Cassie, Akane, Jonathan & Brandon 39% 65% 11% 3- Timary, Zia & Hayden -66.7% 33% 4- Tino, Kyle & Kelsey 31% 41% 5-Delaney, Jenna, Shelby & Brooke -28% 7.7% -27% AVE % Pulse change 56% 5.5% -59% -10.4% 37%

Data Tables Period 3 Code A1 A2 B1 B2 C1 C2 C3 What drug was it? 2mM Caffeine 4mM Caffeine 0.005mM Nicotine 0.05 mM Nicotine Pond Water 1:40 dilution Ethanol 1:20 dilution Ethanol Pulse Change 56% 5.5% -59% 65% -4.7% 11% 37% Soda A= Soda B=

Part II You may have down time while you are waiting on your worms. Do Part II, as time permits and if you have NOT consumed caffeine today. Take a baseline BP (blood pressure) and pulse rate using the BP machine. To do this: be seated Place the cuff snuggly on your arm as shown. Wait until you get a zero display. Press the button to start the reading. Record you initial BP and pulse rate. Obtain a cup of soda and drink it. Note the letter on the side of the cup (A or B) and record it. Wait 10 minutes and take another BP and pulse reading and record.

Part II Which Soda? (A or B) Pulse BP Pre-Soda Post Soda Q8: You will get three numbers from the BP machine, what does each mean? What is considered normal? A8:

Clean-Up Turn off light on stereoscope Wipe off stereoscope stage Place blackworms in detox beaker Rinse off your wax slide with pond water Return all drug samples If you did Part II, please dispose of your cup Return lab packets to front table

Post Lab Questions What is the value of computing the percent pulse rate change? What is the difference seen with different dosages? Why would the pulse change without drug treatment? What is a double blind experiment? You were given the code for each drug… does this count as a double blind experiment? Don’t for get to write a CEC formatted conclusion