Download The Role of Anti-Depressants of Zebrafish

cThe Role of Anti-Depressants of Zebrafish Embryogenesis
Paige O’Donnell
Question:
Given that fluoxetine tends to cause more cardiac defects in human fetuses than the SSRI (serotonin reuptake inhibitor) paroxetine, are these defects dependent on the dose of the drug or the embryonic time of
the drug altogether?
Hypothesis:
Given the varying defects associated with fetal exposure to fluoxetine and paraoxetine, the overwhelming
differences in these effects will depend on the dose of the drugs.
Procedures:
Fertilized zebrafish embryos, they will be divided into sub groups. One group will be the control group while
the others groups will be given different dosages of two SSRIs, paroxetine (Drug A) and fluoxetine (Drug B).
Different doses of anti-depressants will be given to the zebrafish embryos in the following measurements: 10
mg of Drug A or B in 300 mL of zebrafish embryonic medium, 30 mg of A or B in 300 mL of medium, 100 mg
of A or B in 300 mL of medium, 300 mg of A or B in 300 mL of medium, and a control of medium. Give each
dosage to a different group of embryos will be observed and quantified continuously throughout their stages
of embryonic development. These observations will be made under a microscope, using a depression slide to
take a picture, quantification will be made using NIH Image J Software. During the different periods, observe
different aspects of the embryos: zygote period (0-.075 hours)- the size of yolk sacks; cleavage period (1-2
hours) and the blastula period (2-5 hours)- size of yolk sacks and visual amnion; gastrula period (5-10 hours)visual activity of yolk sack; segmentation period (10-24 hours)- size of gut sac; hatching period (48-72 hours)time of hatching; early larval period (72 hours)-the curvature of spine. Since I will be testing what the fine line
between acceptable and dangerous, it will be very important to closely monitor the dosage to quantify an
appropriate teratogenic dose. Throughout the experiment, different set-ups constants such as: nitrites,
nitrate, ammonia, and pH will be kept constant. Once this dosage is empirically determined, NIH Image J
software will be kept to monitor the mortality rate, growth rate, and cardiovascular anomalies caused by the
empirically determined teratogenic dose. At set intervals the zebra fish’s heart will be monitored to see that it
is beating at a normal rate and also to make sure that there are no cardiovascular anomalies since taken. To
calculate the zebra fish’s heart rate, I will count the number beats at a 30 second interval three separate times
and then take the average of that to get in order to get the most accurate count. Additional cardiovascular
defect will be observed and quantified, such as stroke volume and cardiac output. Using the same software,
NIH Image J, the stroke volume will be analyzed by making measurements of the end-diastolic and endsystolic volumes of the ventricle. After attaining these measurements, the cardiac output will be calculated by
multiplying the stroke volume and the heart rate. To measure the survival rate, the dead embryos and each
subsequent day will be counted and sorted; calculating the fraction of surviving larvae at each stage of
development. Once all my data has been collected, I will analyze it to see at what SSRIs defects causes the
most defects or death in the zebrafish embryos. I will have not only my own recordings and observations to
look back upon but also digital pictures that will be taken either periodically or to document an important
finding. Using the different acquired data, and taking a translational approach, these results will then help
determine the ultimate question as to whether the effects of anti-depressants increase a mother’s risk of fetal
cardiovascular defects, and whether these effects are specific SSRI-dependent.
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