.	Kelly Levy             Andrea Wise  Henry Viscardi       Harlandale   School                    High School  Albertson, N.Y.      San Antonio, TX  11507                        78214

 A hormone is a chemical made by one group of cells that is transported through the blood until it reaches a receptor molecule on another organ.  Some processes that are regulated by hormones are growth, development, reproduction, and metabolism.  The organs that make the hormones are called endocrine glands.  Endocrine glands can make one or several hormones.  Each hormone has a specific function that works on one organ or a group of organs.  A hormone has little or no effect on other organs besides the target organ.  Only a specific amount of a hormone will create an effect in a target organ.  This specific amount is called the threshold level.  If the threshold level is not reached then the organ will not respond.

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• Laboratory Activity
• Data Collection
• Graphing

• Anatomy and Physiolgy and Biology students
• 9-12th Grade

Notes to the Teacher: to top

1. Each group should consist of 2 to 4 students
2. Zebrafish need to be maintained for 7-14 days at 14 hour photoperiods to produce embryos.  See ZebraFish web site for details of culturing and maintaining stocks and other lab ideas.
3. Adrenaline solutions : If you purchase a 1:1000 solution of adrenaline from a biological supply house use  10 ml in 90 ml of embryo media to make the 0.0001% solution  and to make the 0.00001% solution take  1 ml of the stock solution and add it to 9 ml of embryo media
4. Embryo Media : 1. Make stock salt solution by mixing 40 g of "Instant Ocean" sea salts with 1 L of distilled water.  Make egg water by mixing 1.5 ml of stock solution with 1L of distilled water to make a final concentration of 60micrograms/ml.
5. This lab can be completed in a 45 minute class period
6. Do not allow students to mix the adrenaline.  Have it prepared in dropper bottles at the front of the class when you begin the lab.

• Zebrafish embryos that are at least 36 hours old so that the heart is formed
• Medicine dropper
• Paper Towels
• Disecting Microscope, Magnifying glass or Compound Microscope which ever you prefer.  You need an objective lense of at least 4 X and no more than 10 X.
• If you are using a compound microscope you  may also may need depression slides to view the embryos
• 3 Petri Dishes
• Adrenaline solutions in dropper bottles :

• Stopwatch

Zebrafish need dechlorinated water which can be created by aging the water for 24 hours before use or you can buying bottled water.  If your area puts chloramine in the water you need to buy bottled water because aging will not remove this substance. (To find out if your water contains chloramine you need to call your local water authority.)  To keep producing embryos for future experiments you will need at least 2 - 10 gallon tanks.  A ten gallon tank can hold anywhere from 25 to 50 fish. One tank for breeding adults, and one for embryos/fry.  Zebrafish will lay eggs but they will cannibalize them.  A dish of marbles should be placed on the floor of the tank.  Fertilized eggs that fall between these marbles will not be eaten.   The eggs can be siphoned or pipetted from the marbles and put into petri dishes of  embryo media. (This can be obtained from a pet store or made, see recipe in teacher information.  For further info, see fish net website )
    To lay eggs the fish need to be on a 14 hour light cycle (14 hrs of light/ 10 hours of dark).  They will lay eggs in the morning when the light is first presented to them.  If you keep them covered you can get them to spawn when you want them to but the photoperiod must remain constant.  The eggs have an 85-100% fertilization rate so once you get them your on your way.  If you feed the fish 2-3 times a day with fish food obtained from your local pet store you can keep them happy and producing eggs every second or third day.  The fry need to eat baby fish food that can be obtained from your local pet store (you are going to get to know the guy at the pet store really well) or you can feed them paramecium until they are 9 days old.  At 9 days you can begin to wean them to brine shrimp.  They should remain separated from the adults until they are 3 months old.  At this point they are ready to begin breeding and will not be eaten by the older fish.
 
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INTRODUCTION

    A hormone is a chemical made by one group of cells that is transported through the blood until it reaches a receptor molecule on another organ.  Some processes that are regulated by hormones are growth, development, reproduction, and metabolism.  The organs that make the hormones are called endocrine glands.  Endocrine glands can make one or several hormones.  Each hormone has a specific function that works on one organ or a group of organs.  A hormone has little or no effect on other organs besides the target organ.  Only a specific amount of a hormone will create an effect in a target organ.  This specific amount is called the threshold level.  If the threshold level is not reached then the organ will not respond.  This lab should help you understand what effect different concentrations of adrenaline will have on the heart rate of  zebrafish.

PURPOSE
    How does adrenaline affect the heart rate of zebrafish?

EQUIPMENT
Zebrafish embryos that are at least 36 hours old
Medicine dropper
Paper Towels
Dissecting Microscope, Magnifying glass or Compound Microscope
Depression Slide(optional)
3 Small Petri Dishes
Adrenaline solutions in dropper bottles
    0.0001% - 1ml needed per student group
    0.00001% -1ml needed per student group
    0.000001% - 1 ml needed per student group
Stopwatch

PROCEDURE
1. Read the lab thouroughly.  Be sure you have read all of the questions that you will be responsible   for answering with this lab.  Be aware of what you are looking for.
2. Place the dish or slide under the microscope and find the embryo's heart.  It is the structure that is under the eye. Here is a diagaram to help you find it:
 
 
 
 

3. Designate who in the group will be the timer, counter and recorder. 
                Timer: ________________________________
                Counter: ______________________________
                Recorder: _____________________________
4. Practice counting the heart beat for 30 seconds.  You should do this 3 times and alternate the  activities so everyone in the group has had a chance to be the timer, counter or recorder.
5. Average the three counts and put that in your data table.
6. Take a petri dish and place the 0.000001% adrenaline solution into it.  Place the embryo in the solution and wait at least one minute for it to take affect.  Using the same proceedure in step 4 count the heart rate and place it in the data table.
8. Locate another embryo on your petri dish and count the heart rate before and after you place it in the dish with 0.00001% adrenaline.  Remember to wait one minute for the adrenalin to take affect.  Make sure to make three counts for each variable.
9. Repeat step 8 with 0.0001% adrealine and record data.
 
 
 

OBSERVATIONS
    Plot your data on a bar graph with heart rate on the y-axis.  Each bar on the x-axis will represent normal and % adrenaline for each concentration.

CONCLUSIONS
1.  What is the target organ in this experiment?
2.  Why do we have to take several trials for each measurement?
3.  What effect do you think adrenaline has on human hearts?
4. Analyse your graph.  At what concentration does adernaline most affect heart rate?  How do you think full strength adrenaline would affect the heart rate of zebrafish heart rates?
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