Influence of Temperature and Neurotransmitters on Heart Rate in crayfish
TA for the Experiment:
Introduction
The cardiovascular system achieves key physiological functions in animals. Particularly, crayfish (Procambarus clarkia), also known as freshwater lobster, have been used to study physiological processes. This is because it is easier to study them in this species than in any other organism. In Crayfish, the circulatory system is open. Blood moves from the heart through the arteries and goes back through the open sinuses. Their heart is located in the cephalotorax, in front of the first abdominal segment, in the dorsal portion.
Basic heart rate beat and rhythm in adult crustaceans depends on a neural output of the cardiac ganglion, which means the heart is neurogenic. Cardiac ganglion rhythm is regulated by the central nervous system, inhibiting or accelerating the cardiac rhythm (Listerman et al. 2000). Heart rate can be measured by creating a low electrical circuit when wires are located on either side of the heart by drilling two small holes on the shell. Impedance of the circuit changes when the heart beats and it is recorded to measure the heart rate (Biology 4510, 2016).
Heart rate, in crustaceans, can be modulated by neurohumoral inputs or by neurotransmitters, as well as by biogenic amines, such as serotonine and dopamine. Epinephrine is a neurotransmitter that excites the heart; on the other hand, acetylcholine inhibits it. Particularly, in crayfish, heart rate is increased by cardioexcitatory peptides, and decreased by the gamma aminobutyric acid (GABA). GABA is an important inhibitory neurotransmitter in mammalian systems (Listerman et al. 2000).
The objective of this laboratory was to determine the effect of the temperature and some neurotransmitters on heart rate in crayfish. The established hypothesis was that if crayfish is treated with low temperature and neurotransmitters, such as serotonin, acetylcholine and epinephrine, the heart rate will change. This is important since studies of heart rate in crayfish can help in physiological studies and give information in other animals, including vertebrates.
Materials and methods
Materials
Impedance Converter.
An Impedance Converter was learned to use. The instrument was turned on and off, by the On-Off-Test. This also allows to determine if the battery is working right. The impedance oscillator was adjusted with precision, 10-turn potentiometer, using the Balance control. Proper operation required the needle around zero.
An AC output was used, and using the AC-Long/Short control. The AC-Long provided a coupling constant of 1 sec, and AC-short of 0.1 sec. To adjust the amplitude of the signal, the Size knob was used. The specimens of crayfish were connected, by electrodes and wires, to the green binding posts on the back of the Impedance Converter in the Input control. The Calib switch was connected to a 0.25 ohm resistor.
Crayfish preparation.
Wires were prepared by cutting two lengths of wire of about 12 inches, burning around 2-3 mm of insulation off of one end of the wires and 1 cm to the other side. Crayfish were secured on a foam pad, and two small holes were drilled through the carapace of the cephalothorax using an 18 g needle. When just a small drop of hemolymph appeared, the drilling was stopped. Wire was placed into the holes, one wire in each hole, and held using a small drop of cyanoacrylate adhesive and small amount of Zip kicker (Figure A.1).
The crawfish was placed in a Tupperware container and 100 mL of freshwater crustacean saline was added. Water temperature was measured. The other ends of the wires were hooked into the input posts on the back of the Impedance Converter.
Figure A.1. Electrode puncture site. Place were cephalothorax was drilled and wire were inserted. (Biology 4510, 2016)
Heart Rate measurements.
Baseline Heart Rate. For the baseline heart rate, the Tupperware container was covered with foil. When the crawfish was quiet, the heart rate started to be measure by clicking the Start control. The heart rate was measured for 5 to 10 minutes or until it was steady. Finally, measurement was stopped by clicking the Stop, and start and end heart rate was registered.
Stressed Heart Rate. For stressed heart rate, the same procedure was done, but by uncovering the container.
Serotonin. To determine the influence of serotonin on heart rate, one milliliter of 1mM Serotonin solution was added to the container and the final concentration of the neurotransmitter was calculated. The container was covered, and after 5 min, the trace was started and recorded for 5 to 10 minutes. Measurement was stopped and serotonin heart rate trace was annotated.
Acetylcholine. Freshwater Crustacean Saline was replaced in the container, and one milliliter of 1 mM Acetylcholine solution was added, and final concentration was calculated. After 5 min of covering the container with foil, the measurement was done as was explained before, for 5 to 10 minutes. Start and end of acetylcholine heart rate trace was recorded.
Epinephrine. One milliliter of Epinephrine 1 mM was added to the container having new Freshwater Crustacean Saline. The container was covered and after 5 min readings of epinephrine, heart rate trace was reported. Holding water was replaced, as was done before, by adding 100 mL of Freshwater Crustacean Saline.
Temperature. Covered container with having the crawfish was placed on the surface of ice and waited for 5 minutes. Water temperature was carefully measured. The trace was started and recorded for 5 to 10 minutes. After finishing the experiment, the container was removed from the ice.
Results
Heart rates were determined in crayfish using an impedance converter. Crayfish was secured on a foam pad and wires were placed on either side of the heart, creating a low voltage electrical circuit including the hemolymph and heart tissue. Heart beats were measured by changes in the impedance of the circuit.
Three different neurotransmitters (serotonin, acetylcholine and Epinephrine), and cold temperature were applied to determine the influence on heart rate. In doing this, after applying the treatment for five minutes, heart rate was measured. All neurotransmitter final concentrations were 0.01 mM, since 1 mL of 1mM concentration was added to 100 mL of Freshwater Crustacean Saline.
BPM: beats per minute
Comparisons between all treatments are presented in figure A.2. Differences were found in all treatments when compared to the baseline and stressed conditions. Serotonin, acetylcholine and epinephrine showed higher values than the baseline. In contrast, cold (temperature treatment) was lower than the baseline.
Figure A.2. Comparison between treatments for heart rate measurement in crayfish. All neurotransmitters are at final concentration of 0.01 mM. Heart rate is express in beats per minute (BPM).
Discussion
Rapid changes in heart rate are the response to external stimuli and substances, possibly having an inhibitory or excitatory effect. In that way, heart rate can increase or decrease in response to external stimuli.
Stressed animals show higher heart rate than the baseline. Aquatic animals, particularly crayfish, become more active when they are stressed for being in low water levels. This reaction occurs because they are trying to find a new source of water or a similar environment where they live. Normal heart rate in crayfish is between 60 BPM and 120 BPM at 24ºC. This is in accordance to the baseline in this study, which was around 80 BPM.
When neurotransmitters, such as serotonin, acetylcholine and epinephrine were used, heart rate changed in comparison to the baseline. The same observation was found with the temperature.
Serotonin is an endogenous neurotransmitter. The increase of heart rate was evident, when compared to baseline and stressed measurement. Serotonin influences social behavior of the decapods, which is related to serotonin receptors. Studies have shown the increase of heart rate in crayfish when the level of serotonin is progressively increased from 100 nM to 10 µM (Kawai et al. 2016). A concentration of 0.01 mM was used in this study. In crustaceans, the degree of aggressiveness is correlated to serotonin levels in hemolymph. This can be associated to an increase in heart rate.
A similar response was found for acetylcholine and epinephrine: a higher heart rate was found than baseline. When compared to the stress treatment, a similar value was found. In crustaceans, acetylcholine speeds up the heart rate, contrary to vertebrates, where it slows down the heart rate, via vagus nerve (Olshansky et al. 2008). On the other hand, epinephrine and adrenaline increases the heart rate in both vertebrate and invertebrates.
Temperature also affected heart rate in a positive correlation: when temperature decreased, heart rate also decreased. In addition, crayfish survive for at least a week when temperature drops from 21 ºC to 5 ºC (Chung et al. 2012).
Conclusion
Heart rate is influenced by neurotransmitters and temperature. Serotonin, acetylcholine, and epinephrine excite heart rate, increasing it. This is contrary to temperature, which reduces heart rate as temperature is reduced. Low standard deviation demonstrated a good performance in the experiments and precision of the equipment.
Literature cited
Biology 4510: Laboratory Manual. (2016). Crayfish Cardiac Physiology. Adapted from Tullis A.
Chung YS, Cooper RM, Graff J, Cooper RL (2012). The Acute and Chronic Effect of Low Temperature on Survival, Heart Rate and Neural Function in Crayfish (Procambarus clarkii) and Prawn (Macrobrachium rosenbergii) Species. Open J Mol Int Physiol 2: 75-86.
Kawai T, Faulkers Z & Scholtz G (2016). Freshwater Crayfish: A Global Overview. CRC Press, New York.
Listerman LR, Deskins J, Bradacs H & Cooper RL (2000). Heart rate within male crayfish: social interactions and effects of 5-HT. Comp Bioch Physiol Part A 125: 251-263.
Olshansky B, Sabba HN, Hauptma PJ, Colucci WS (2008). Contemporary Reviews in Cardiovascular Medicine Parasympathetic Nervous System and Heart Failure. Pathophysiology and Potential Implications for Therapy. Circulation 118:863-871.
