Investigating the Effects of Agonist and Antagonist Drugs on Daphnia Heart Rate

Investigating the Short-term Effects of Commonly Known Agonist and Antagonist Drugs on the Heart Rate of Daphnia Abstract Although Daphnia are invertebrates, evidence suggests that Daphnia and vertebrates have similar heart structures and are regulated in a comparable manner. This experiment aims to test agonist and antagonist drugs – whose effect on humans are known – on Daphnia, identifying its effects on the organism and comparing it to those on humans. Previous scientific research on the effects of the aforementioned drugs on human heart rates serves as the hypothesis for the effects of the drugs on Daphnia. The basal heart rates of the Daphnia were measured using the magnification of a compound microscope. The Daphnia were then exposed to caffeine, lidocaine, epinephrine, and nicotine individually for 1 minute, recording the heart rate again afterwards. Results show that caffeine caused an increase in heart rate in the Daphnia while lidocaine, epinephrine, and nicotine caused a decrease. These results support half of the stated hypotheses while refuting the other half. Future studies can be conducted to improve the accuracy of this experiment, providing more concrete conclusions. Introduction Daphnia, commonly known as water fleas, are crustaceans and are thus invertebrates. They are commonly used for ecotoxicological testing, as they are small and easy to rear in lab (Peake et. al 2016). Because they are invertebrates, one would think that the Daphnia would have different heart structures from those of vertebrates. However, evidence shows that the Daphnia heart is myogenic, just like vertebrate hearts (Bekker & Kirjgsman 1951). In its simplest form, vertebrate hearts have five major components in their conduction system, which makes use of an orderly sequence of cardiac cell contractions: the sinoatrial node (or pacemaker), the atrioventricular node, the atrioventricular bundle, bundle branches, and the Kay Htut conduction myofibers. The pacemaker cells contract to create rhythmic impulses that directly control the heart rate, and the rate and strength of these contractions are regulated by sympathetic and parasympathetic nerves of the autonomic nervous system. (MedlinePlus 2018). The sympathetic nervous system is activated in stressful situations while the parasympathetic nervous system allows organisms to rest and digest (Blessing & Gibbins 2008). Both pathways produce neurotransmitters that can bind to either ionotropic or metabotropic receptors, both of which regulate the opening and closing of ion channels. This can either activate or inhibit cellular responses. Neurotransmitter binding can be regulated by a wide variety of drugs that influence the ability of receptors to bind to their endogenous ligand. An agonist drug fully activates the receptor it binds to, mimicking the effect of a natural ligand in the body. On the contrary, an antagonist drug blocks the natural ligand from binding to its receptor (Weir 2016). The four agonist and antagonist drugs studied in this experiment – caffeine, lidocaine, epinephrine, and nicotine – have been researched extensively and their effects on human heart rate are known. Caffeine is a receptor antagonist that has been shown to increase heart rate in humans (Geethavani et. al 2014). Lidocaine is an antagonist and fixes irregular heart rates by causing the heart to slow down (Courseault 2017). Epinephrine is both a hormone and a medication that can be used to treat multiple illnesses, such as anaphylaxis and cardiac arrest. It is known to increase heart rate in humans (Klabunde 2016). Nicotine is a stimulant that acts as a receptor agonist, increasing heart rate (Benowitz & Gourlay, 1997). In this lab, the effects of specific agonist and antagonist drugs that were previously mentioned are studied on Daphnia. Understanding the impact drugs commonly used in humans have on animals can be extremely useful. If another species reacts to a drug as a human would, it suggests that the species may have similar cell signaling pathways as humans. Not only would this knowledge be useful for scientific research, but for clinical Kay Htut trials that would normally be costly and possibly dangerous if conducted on humans. Comparing the effects of the drugs in Daphnia to the effects of the same drugs in humans can allow us to draw conclusions about the cell signaling pathways of Daphnia and humans. Using previous research, it is expected in this experiment that caffeine, epinephrine, and nicotine would increase heart rates in Daphnia whereas lidocaine would decrease heart rates. Materials and Methods A small drop of Detain solution was first placed in the well of a depression slide. One Daphnia specimen was transferred from the culture on the supply bench to the depression slide using a pipette. The organism was positioned such that it becomes immobilized in the Detain solution. The Daphnia was examined under a compound microscope using the 10x objective to locate and identify the dorsal heart. The basal heart rate of the Daphnia was determined by observing the heart rate under the microscope and counting the number of beats in a 15 second time period, repeating to obtain a total of two heart rates. The data was then normalized to beats per minute and the two basal heart rate measurements were averaged together. The slide was immediately removed from the microscope to allow the slide to cool, preventing further harm to the Daphnia. A reagent spatula was used to gently lift the Daphnia out of the Detain resin and place it into a depression well containing fresh water. The organism regained mobility in the water as it washed off the resin. One drop of a chosen drug was placed into a depression well, and the same Daphnia was placed into the drug solution using a spatula. After one minute, the Daphnia was placed onto the slide once again and then immobilized with a drop of Detain resin. Using the same method as above, the average drug-induced heart rate of the Daphnia was found. The Daphnia was then discarded in a small culture container marked “Used”. Kay Htut Twelve different Daphnia (three for each of the four tested drugs) underwent these procedures. The drugs tested were caffeine, lidocaine, epinephrine, and nicotine. Results Caffeine Average Heart Rates 180 Heart rate (beats/minute) 160 140 120 100 80 basal 60 drug-induced 40 20 0 1 2 3 Trial Figure 1. The average heart rates of Daphnia before and after exposure to Caffeine. Three Daphnia (n = 3) were examined by two different people to obtain two basal heart rates, which were averaged together. The Daphnia were then exposed to one drop of Caffeine for one minute. Their heart rates were subsequently measured twice and averaged together. Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Kay Htut Caffeine Average Heart Rates (Class Data) Heart rate (beats/minute) 400 350 300 250 200 basal 150 drug-induced 100 50 0 1 2 3 4 5 6 7 8 Trial Figure 2. Class results of the average heart rates of Daphnia before and after exposure to Caffeine. The averaged basal and drug-induced heart rates from 7 other groups were obtained using the same methods described in Figure 1. The heart rates of the three different Daphnia were averaged together for each group data and combined with the averaged data from Figure 1 to produce this figure (n = 24). Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Lidocaine Average Heart Rates (Group Data) Heart rate (beats/minute) 140 120 100 80 basal 60 drug-induced 40 20 0 1 2 3 Trial Figure 3. The average heart rates of Daphnia before and after exposure to Lidocaine. Three Daphnia (n = 3) were examined by two different people to obtain two basal heart rates, which were averaged together. The Daphnia were then exposed to one drop of Lidocaine for one minute. Their heart rates were subsequently measured twice and averaged together. Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Kay Htut Lidocaine Average Heart Rates (Class Data) Heart rate (beats/minute) 400 350 300 250 200 basal 150 drug-induced 100 50 0 1 2 3 4 5 6 Trial Figure 4. Class results of the average heart rates of Daphnia before and after exposure to Lidocaine. The averaged basal and drug-induced heart rates from 5 other groups were obtained using the same methods described in Figure 3. The heart rates of the three different Daphnia were averaged together for each group data and combined with the averaged data from Figure 3 to produce this figure (n = 18). Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Epinephrine Average Heart Rates (Group Data) Heart rate (beats/minute) 200 180 160 140 120 100 80 60 40 basal drug-induced 20 0 1 2 3 Trial Figure 5. The average heart rates of Daphnia before and after exposure to Epinephrine. Three Daphnia (n = 3) were examined by two different people to obtain two basal heart rates, which were averaged together. The Daphnia were then exposed to one drop of Epinephrine for one minute. Their heart rates were subsequently measured twice and averaged together. Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Kay Htut Epinephrine Average Heart Rates (Class Data) Heart rate (beats/minute) 350 300 250 200 150 basal 100 drug-induced 50 0 1 2 3 4 5 6 7 8 Trial Figure 6. Class results of the average heart rates of Daphnia before and after exposure to Epinephrine. The averaged basal and drug-induced heart rates from 7 other groups were obtained using the same methods described in Figure 5. The heart rates of the three different Daphnia were averaged together for each group data and combined with the averaged data from Figure 5 to produce this figure (n = 24). Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Nicotine Average Heart Rates (Group Data) Heart rate (beats/minute) 350 300 250 200 150 basal 100 drug-induced 50 0 1 2 3 Trial Figure 7. The average heart rates of Daphnia before and after exposure to Nicotine. Three Daphnia (n = 3) were examined by two different people to obtain two basal heart rates, which were averaged together. The Daphnia were then exposed to one drop of Nicotine for one minute. Their heart rates were subsequently measured twice and averaged together. Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Kay Htut Nicotine Average Heart Rates (Class Data) Heart rate (beats/minute) 350 300 250 200 150 basal 100 drug-induced 50 0 1 2 3 4 5 6 Trial Figure 8. Class results of the average heart rates of Daphnia before and after exposure to Nicotine. The averaged basal and drug-induced heart rates from 5 other groups were obtained using the same methods described in Figure 7. The heart rates of the three different Daphnia were averaged together for each group data and combined with the averaged data from Figure 7 to produce this figure (n = 18). Error bars were produced in Excel using the standard error. Averages were also computed through Excel. Discussion The purpose of the lab was to determine the effects of certain drugs on the heart rate of Daphnia. The obtained results support the hypothesized effects for half of the drugs while refuting them for the other half. Previous scientific research has found that caffeine, epinephrine, and nicotine increase heart rate, while lidocaine decreases heart rate. The results for caffeine support the hypothesis for the most part. While all trials of the group data show an increase in heart rate, 3 of the 8 groups in figure 2 show a decrease in heart rate. The error bars show that even considering the standard error, the drug-induced heart rates would still generally be higher than the basal heart rates. The lidocaine data shows a strong consensus in supporting the hypothesis, seeing as how all trials of the group data and all groups of the class data show a decrease in heart rate. Error bars for the group data show small standard error and thus, high confidence levels. As for epinephrine, while it is stated to increase heart rate Kay Htut (Klabunde 2016), this experiment showed greatly conflicting results. All trials of the group data showed decreased heart rates, as did all but one group in the class data shown in figure 6. Additionally, the data for nicotine do not reinforce the scientific fact that nicotine increases heart rate (Benowitz & Gourlay 1997). Experimental nicotine data show a general disagreement for the hypothesis, as the trials in figure 7 all show decreasing heart rates and only half of the groups in figure 8 show increasing heart rates. While the data for caffeine and lidocaine support previous research, there are discrepancies in the data for epinephrine and nicotine. It is important to note that the experimental data conflicting with previous scientific research are those that show decreased heart rates when they were expected to show increased heart rates. These errors could originate from the fact that the Daphnia are exposed to heat while being observed on the microscope. Excessive heat exposure harmfully affects and can potentially kill Daphnia (Woodham et. al 2018), so if the Daphnia is left on the microscope for too long without turning off the lamp, its heart rate will gradually decrease until it is dead. Some Daphnia also died directly after being exposed to the drug. It is not known whether this is because the drug dosage was too much or if the process of extracting the Daphnia from its environment was too stressful for the organism. This made the Daphnia difficult to work with during the extraction processes. Additionally, heart rates were measured through manual observation and counting, incorporating human error. The number of heart rates obtained and the amount of trials taken are also not enough to draw concrete conclusions from. These sources of error make the experimental data not entirely reliable. Future studies could further explore the correlation between drug dosage and the health of the Daphnia. If the ideal dosage of each drug is found to prevent the Daphnia from death before observation while simultaneously showing the effects of the drugs, the experiment may be repeated with greater accuracy. Furthermore, using an alternative method Kay Htut of measuring the heartbeats of the Daphnia would limit human error. A different microscope could also be used that does not expose the Daphnia to too much heat.