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Daphnia Experiment: Measuring Heart Rate Effects of Caffeine & Alcohol, Lecture notes of Pharmacology

University of the Arts London (UAL)Pharmacology

In this two-week lab experiment, students will learn how to measure the heart rate of daphnia, a small water flea, and investigate the effects of caffeine and alcohol on heart rate using experimental design, data acquisition, and statistical analysis. Skills developed include experimental design, data analysis, and use of microscopes and pipettes.

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HEART RATE PHARMACOLOGY OF THE WATER
FLEA, Daphnia (Kingdom Animalia; Phylum Arthropoda; Class Crustacea)
Summary of the Lab:
Week 1: Learn how to measure the heartbeat rate in a small crustacean, Daphnia. Design an
experiment to test the effects of either caffeine, or alcohol on the heart beat rate in
Daphnia. Form a hypothesis and design an experiment to test that hypothesis concerning the effect
of a substance on heartrate in Daphnia.
Week 2: Perform the experiment you designed in Week 1.
Skills you will begin learning in this lab:
1. How to formulate a testable hypothesis
2. Experimental design, including controls and treatments
3. Data acquisition, summary, and analysis
4. Computer graphing and statistical analysis
5. light microscopy (stereo dissection microscopes)
6. simple dilution; serial dilution
7. pipetting using automatic pipettes
8. dose-response curves
9. cooperative working with your group
10. scientific writing
Introduction
References: Barnes, Invertebrate Biology 5tb ed. (available in lab).
We are constantly being exposed to drugs in
our environment by inhalation, ingestion, skin
contact, etc. This exposure can either be
deliberate or inadvertent. Some of these drugs
affect important metabolic functions. The study
of the affect drugs have on metabolism is part
of the science of Pharmacology. For this
experiment, we will investigate the effects the
drugs caffeine and alcohol (or another drug of
your choice) on heart rate. Often scientists are
interested in the effect of drugs on mammalian
physiology, but studies with mammals (and
especially humans) can be problematic for
many ethical and logistical reasons. We will
therefore use a small crustacean called
Daphnia as an experimental model in which to
begin this investigation.
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HEART RATE PHARMACOLOGY OF THE WATER

FLEA, Daphnia (Kingdom Animalia; Phylum Arthropoda; Class Crustacea)

Summary of the Lab:

Week 1: Learn how to measure the heartbeat rate in a small crustacean, Daphnia. Design an experiment to test the effects of either caffeine, or alcohol on the heart beat rate in Daphnia. Form a hypothesis and design an experiment to test that hypothesis concerning the effect of a substance on heartrate in Daphnia. Week 2: Perform the experiment you designed in Week 1. Skills you will begin learning in this lab :

  1. How to formulate a testable hypothesis
  2. Experimental design, including controls and treatments
  3. Data acquisition, summary, and analysis
  4. Computer graphing and statistical analysis
  5. light microscopy (stereo dissection microscopes)
  6. simple dilution; serial dilution
  7. pipetting using automatic pipettes
  8. dose-response curves
  9. cooperative working with your group
  10. scientific writing

Introduction

References: Barnes, Invertebrate Biology 5tb ed. (available in lab). We are constantly being exposed to drugs in our environment by inhalation, ingestion, skin contact, etc. This exposure can either be deliberate or inadvertent. Some of these drugs affect important metabolic functions. The study of the affect drugs have on metabolism is part of the science of Pharmacology. For this experiment, we will investigate the effects the drugs caffeine and alcohol (or another drug of your choice) on heart rate. Often scientists are interested in the effect of drugs on mammalian physiology, but studies with mammals (and especially humans) can be problematic for many ethical and logistical reasons. We will therefore use a small crustacean called Daphnia as an experimental model in which to begin this investigation.

The Model Organism: Daphnia (Kingdom Animal; Phylum Arthropoda; Class Crustacea) We picked Daphnia , commonly called a 'water flea', for several reasons. The heart is relatively easy to see because the organism is transparent. Also, Daphnia are small and easy to obtain in large numbers. Finally, since we will immerse the organism in a solution of pond water mixed with a drug, the animals will respond quickly to this environmental change (i.e....we don't have to wait for the circulatory system to transport an injected drug throughout the critter s body). Daphnia are small fresh water crustaceans that are completely transparent, thus making observation of heart beat rate and other internal functions easy. Daphnia uses its large second antennae to swim in a characteristic jerky style. They have compound and simple eyes similar to those found in insects and the eyes serve to orient the swimming of the animal. They are filter feeders with a complex gut and much of the motion you will observe in Daphnia is feeding. They can feed on plankton (floating protists/insect larva/etc. and some species can even live solely on ingested bacteria. Most common species of Daphnia have separate male and female sexes and the females carry the developing embryos within their shell on their dorsal side. Some species are parthenogenic and lack males; these species develop solely by asexual reproduction. The heart has two chambers and its beating is rapid (often >100-160 bpm at room temperature) and quite distinctive. In highly aerated water, the blood is transparent. When grown in stagnant water where the level of dissolved oxygen is low, the animal produces large amounts of hemoglobin in its blood giving the animal a reddish pink hue.

Lab Outline

Week 1: During lab the first week you will learn how to measure the heart rate of Daphnia and how to apply drugs (see protocol that follows.) Once you have obtained crude estimates of baseline heart rate, consider what aspects of your technique must be carefully controlled so as to achieve reproducible results. When you have mastered the technique and feel confident of maintaining constant experimental' conditions, design an experiment which will yield a estimate of heart rate when no drugs are applied (CONTROL), and at least 3 concentrations of the drug (TREATMENT). Discuss your experimental design with your instructor to get feedback and suggestions. Week 2: During week 2 of this lab your group will carry out the experiment you designed and have since refined. The materials you need will be available in lab and you need not wait for your instructor to say start. Get as much data as you can. Basic procedure for measuring heart rate in Daphnia :

  1. Since the heart beats so rapidly that it is difficult to count, we have found that chilling the Daphnia to approximately 10°C gives a countable rate. We will call this rate our BASELINE or CONTROL heartrate.
  2. To achieve this rate, the Daphnia have been kept in 10°C pond water for >60 minutes. Obtain a single animal with a soft plastic pipette and put it in a large drop of cold pond water on a pre-chilled slide. Then, transfer the slide to the controlled temperature chamber on the stage of a dissecting microscope. You may want to put a piece of black construction paper under the chamber to increase the contrast. Angling your light source from one side usually helps increase the contrast as well.

h) How would you do a dose-response curve? What will it tell you? Could you use the curve to predict the outcome of a follow-up experiment? i) What 'dangers' are inherent in extrapolating from a dose-response curve? Writing up your results: This write-up will require an Introduction that will force you beyond our text as a sole reference. Use the library to obtain some BASIC background information that may help you answer questions such as : What are these drugs and what do we know about them? Why did you select the particular drug you chose? Is this drug important in our society? What do we know about this drug's effect on animals? (in general?) Why should they affect heartrate? Is heart rate a good indicator of drug activity? Do we know the mechanism of action for this drug that lets us explain how it effects, or should affect, heart rate? Consider what difference(s) exist between a mammalian heart and a crustacean heart. Will this effect the action of the drug? You will find little or nothing specific to the effect of a drug on Daphnia per se in our library, so consider any article as relevant that deals with the action of the drug, especially if that action affects heart activity either directly or indirectly. You should have at least two references from the primary literature. The publications listed below are the three most pertinent ones that we have been able to find. They can be found in the lab and are to remain there, except for brief trips to the photocopier. We have also placed in the lab a couple of very general invertebrate zoology texts from which you may be able to glean some basic, but useful, information on Daphnia. In addition to these sources, we want you to find at least two other sources that pertain to your experiment. At least one of these should be from the primary literature Foster, R. 1997. A stroboscopic method to investigate the effect of caffeine on Daphnia heart rate. Journal of Biological Education 31 : 253-255. (also available in McIntyre Library) Ruediger J.P., Colmorgen M., Hueller S., Tyroller F., Dietmar Z. 1997. Circulation and respiratory control in millimitre-sized animals ( Daphnia magna, Folsomia candida ) studied by optical methods. Journal of Comparative Physiology B 167 : 399-408. McMahon, BR, Wilkens JL, 1983. Ventilation, Perfusion, and Oxygen Uptake. In : Biology of Crustacea, Vol. 5, Internal Anatomy and Physiological Regulation (LH Mantel, ed.). Academic Press, New York.