The purpose of this lab was to study the behavior of action potentials in earthworms. Earthworms are ideal for the study of action potential conduction velocity because of their simple structured, easily measured bodies. Earthworms have two giant fiber nerve systems, lateral and medial, in which they conduct velocities that are easily measured through electrode manipulation. To indicate the action potential, axon myelination and diameter size are important factors. According to some studies, a larger axon diameter has resulted in a faster action potential in the axon. Most importantly, tripling the myelin thickness increases the conduction velocity by 3 times, whereas tripling the axon diameter resulted in an increase to the conduction velocity by square root of 3, or 1.7 times (CCNY BIO. 85). The hypothesis made for this study was that the medial giant nerve, MGN, would have a more rapid conduction velocity speed than the lateral giant nerve, LGN, due to a thicker myelin. In this experiment, using a Spike Recorder, the conduction velocity of earthworms were measured from both anterior and posterior ends. As hypothesized, the mean conduction velocity was significantly higher in the MGN than in the LGN.
In this lab, the behavior of action potentials were studied in earthworms. Action potentials are the electrical currents that are made by our nerves when we receive a stimulus, allowing us to feel things and make movements. The medial and lateral giant nerves both transmit various sensory information from various parts of the worm’s body.
In order for the occurence of an action potential, a threshold value must be reached by the stimulus. Action potentials happen when the sodium channels are activated on a the membrane causing a high permeability to sodium ions, which causes the membrane to depolarize. When potassium channels open and sodium channels close, the membrane begins to repolarize, allowing it to return to its resting potential. (Russell et al. 2016). During this process, only one stimulus can create an action potential at a time and a second one cannot be generated with any stimulus; this is known as the refractory period.
During this lab, the main focus was to view the action potential of the worms and to calculate the conduction velocities of both the Medial and Lateral axons. Although action potentials usually only travel in one direction, since we are manually stimulating the worms, the action potential can be measured in both directions with modifications to the electrodes. Keeping into mind that the LGN transmits sensory information from the posterior end to the anterior end, while the MGN transmits sensory information form the anterior end to the posterior end, allowing for easily measured muscle contractions. The class hypothesized that the conduction velocity of MGN would conduct a faster action potential than the LGN, due to the MGN having a larger axon diameter. Typically, as axon increases its diameter, its myelin thickness also increases. Perhaps the MGN has a thicker myelin sheath as well (Backyard Brains. 2019).
Materials and Methods
In this experiment, the materials that will be the Lumbricus terristrius, known as an earthworm. We will also be using 10% ethanol solution, the Faraday cage and 2 channel SpikerBox, a piece of balsa wood or thick cork, USB cable, Laptop with Spike Recorder software, ruler, tape and marker.
In all experiments in this report, earthworms were provided to conduct readings on. The healthy earthworms were placed into 10% ethanol solutions for 2-3 minutes each so that they could be anesthetized. They were originally supposed to be left in this ethanol solution for 7-10 minutes; however, they were not left in there for that long because too much anesthesia would cause nerves to not fire, and too little anesthesia would cause movement during the experiment, resulting in muscle electrical activity, which will mess with the small neural electrical signals that are meant to be obtained (CCNY Bio Lab Manual. 73). Shortly after the earthworm stopped moving, they were placed into a beaker of distilled tap water for a few seconds just to rinse their bodies off. The earthworm was then placed onto a piece of balsa wood, which was covered in saran wrap for preservation, dorsal side up and was kept moistened throughout the experiment by wetting a piece of tissue and delicately touching their skin, making sure to avoid the upcoming electrodes. The balsawood was then placed into a Faraday cage, which was easily accessible from anterior and posterior ends. After that, with the two-channel SpikerBox, the 3 electrodes were placed, slightly off the centerline (to avoid piercing intestines or ventral nerve cord), into the anterior end of the worm. The red and white electrode were 0.5 inches apart and the white and black were 1.5 inches apart. The electrodes were plugged into the Neuron SpikerBox Pro and the USB cable into a PC. Open the Spiker Recorder software and click on the USB symbol to pair with the Neuron Spiker Box. Next, press the record button and using a plastic or wooden probe, gently tap the anterior end of the worm. Once several spikes were obtained, they were recorded. This procedure we then repeated when trying to obtain recording from the posterior end. Once completed, the worms were returned back into their soil. They make look really tired and worn out but the worms are able to handle a lot and recover well from the experiments.
Figure 1. This chart represents the average measurements of the lateral and medial giant fiber conduction velocities in the earthworm; along with the standard deviation.
In Figure 1, the data represents the means and standard deviations of the medial and lateral giant nerve fibers. The mean conduction velocity of the MGN, 14.5 m/s, with a standard deviation of 3.2, was significantly higher than that of the LGN, 8.52 m/s, whose standard deviation was 1.98.
T-Critical for 95% Confidence Level
Degrees of Freedom
97.5%< x <99%
Table 1. This chart represents the statistical analysis between the average of both groups, MGN and LGN.
Table 1 represents the statistical analysis that was conducted using the mean, standard deviation and sample sizing from both MGN and LGN. According to the table, T-Calc had a value of 5.71. There were 2 sample sizes of 13, leading to a value of 24 degrees of freedom. With a 5% confidence level/ p-value range, it is shown that the t-critical value, 1.71 was less than the t-calc value. This means that our data is significant, leaving us with the confidence level of being >97.5% but <99%.
In conclusion, it was found that the MGN, anterior end, conduction velocity was indeed faster than the LGN, the posterior end. Our hypothesis that the MGN would transport a faster action potential than the LGN has been proven true. This hypothesis is supported by both results provided, along with background information, since the data is statistically significant, which is shown by the error bars in Figure 1. Also, t-calc is greater than t-critical with above a 95% confidence level.
In every experiment, there are always a vast amount of opportunities to cause errors, whether human or experimental. Many errors could have derived from using the Spike Recorder. A person conducting this experiment might have a heavy hand, so their force could have an affect on the action potential because of a greater stimulus to the worm unlike someone with a more delicate touch. Another possible error could be with the measurements of the distance and the placement of the electrodes. Maybe a group could have been reading the data backwards, so while doing anterior readings, their electrodes were placed in the order for reading posterior spikes. Some other ways to go about this experiment would be to try different organisms to experiment on.
Shannon, K. M., Gage, G. J., Jankovic, A., Wilson, W. J.,
Metabolic Regulation Differences Between a Thyroidectomized Mouse and in a Hypophysectomized Mouse
Taking a look at the thyroid gland, as part of the hypothalamic-pituitary-thyroid axis, the two thyroid hormones it releases, triiodothyronine (T3) and thyroxine (T4), play an important role in regulating the body’s metabolism. Metabolism is the summation of both anabolic and catabolic chemical reactions that occur in the cells of the body. The more thyroid hormones that have the potential to bind to specific cells, the higher the rate of metabolism that those cells will undergo. Of course, the amount of these hormones needs to be homeostatically regulated under a negative feedback system by the hypothalamus.
Through a humoral control system, the hypothalamus regulates the thyroid hormone concentration in the blood through a specified set point. If either the concentration of the thyroid hormones is too low or too high in the blood, the hypothalamus will increase or decrease, respectively, its release of thyrotropin-releasing hormone (TRH) to the adenohypophysis. Subsequently, the adenohypophysis has cells that respond to specific hormones that the hypothalamus releases, in this case it is TRH. When TRH is present, the anterior pituitary will release more thyroid-stimulating hormone (TSH) which will arouse the thyroid gland to release thyroid hormones. The aim of this study is to observe and interpret the effects on the metabolic activities of mice that either have the absence of the thyroid gland or the absence of the pituitary gland when exposed to three injections that contain T4, TSH, and propylthiouracil (PTU). Each of those substances possesses an effect on either the hypothalamus/pituitary or the thyroid gland.
Measuring the Baseline Metabolic Rate (BMR) Through Oxygen Consumption
This experiment utilized three mice with identical age and sex and of similar weight. One of the mice underwent thyroidectomy, the other underwent hypophysectomy, and the third untouched to become the control subject. They were kept is three separate cages, consuming the same amounts of food and water at the same time each day. A week after baseline measurements, each mouse was individually placed in a sealed chamber that contained a scale for one minute.
This chamber contained two tubes connected. One tube was accessible to the outside air, which will be open when the subject is not being measured, and closed when the subject is being measured, while the other tube ran through a T-shaped pipe, which directed air flow into one of the two passageways. One of the dividing tubes led to a U-shaped manometer filled with water. The manometer measures the oxygen consumption by offsetting the water level of one side of the U to the other side. The other dividing tubes led to a syringe filled with air. This syringe was used to pump air into the tubing system towards the manometer in order to level the water. The amount that the syringe measures when the manometer reaches the original leveled state is the amount of oxygen that the test subject consumed in that one minute.
Each mouse was untreated and then measured for their weight (in kilograms) and oxygen consumption (in milliliters) to establish baseline values. Afterward, each mice was injected with T4 then remeasured for their weight and oxygen consumption. This was repeated for the administration of TSH and again for the administration of PTU after all three of the mouse had returned to near their standard BMR. Calculations must be made using the oxygen consumption and weight recorded for each mouse after each of the trials in order to calculate the BMR of each mouse in the experiment. The equation is as follows: milliliters of oxygen consumed per hour divided by the weight of the mouse in kilograms. Consequently, the BMR values will be expressed in ml O2/hr/kg.
The Hypothalamic-Pituitary-Thyroid Axis
As mentioned, through a homeostatically regulated negative feedback system, the hypothalamus is the main control center in regulating thyroid hormone release. Nillni (2010) states that the afferent neurons that play an important role in informing TRH neurons in the hypothalamus are the catecholamine neurons, which originates in the brain stem, and the arcuate nucleus of the hypothalamus (ARC). Peripheral neuronal signals that are stimulated for signs of starvation or illness cause a response from the ARC while temperature decrease sensed by peripheral afferent temperature receptors cause a response from the catecholamine neurons. When stimulated, these afferent connections signal the hypothalamus TRH neurons to release more TRH.
Going into context with the negative effects of thyroid hormones on the TRH neurons, in another study by Mariotti and Beck-Peccoz (2016) stated that specialized ependymal cells identified as tanycytes, located along the blood-brain barrier of the choroid plexus as well as the third ventricle, withdraw T3 and T4 from the bloodstream across the blood-brain barrier and from the cerebrospinal fluid (CSF). The T4 will then be converted into the more bioactive hormone T3, using the type 2 iodothyronine deiodinase (D2) enzyme – which the differences between the two thyroid hormones will be discussed later. As a steroid hormone, T3 is a hydrophobic hormone that binds mainly on intracellular receptors meaning that it can usually pass through the target cell’s phospholipid bilayer, but in order to enter the TRH neurons it requires a series of active transporters, mainly two: organic anion transporting polypeptide (OATP) and the monocarboxylate transporter type 8 (MCT8). How TRH neurons work as their own differentiated neuron is in the concept of its TRH production. Along the cell’s genome lies the promoter region for the creation of mRNA strands to synthesize TRH to release to the adenohypophysis. Normally, there is an abundance of CREB (cAMP response element binding protein), a transcription factor that, in this case, increases the transcription of the TRH gene into mRNA for translation. T3 negatively affects this process by binding into thyroid hormone receptor/T3 retinoid X receptor complex which ultimately lowers the CREB concentration, lowering the transcription of the TRH gene.
As TRH is synthesized by the TRH neurons, the TRH neurons release the hormone through their axon terminals in the median eminence, which is located on the superior side of the adenohypophysis, and into the blood circulation of the adenohypophysis. Specialized cells named thyrotrophs own TRH receptors for the TRH neuropeptide hormone. These TRH receptors are G-protein-coupled receptors. As a result of the G-protein activation, the alpha subunit from the G-protein then activates the protein lipase C enzyme, which, in effect, breaks down phosphatidylinositol 4,5-bisphosphate (PIP2), which is rich in the cell cytosol as an important substrate to many signaling proteins, to inositol trisphosphate and diglyceride. Inositol triphosphate then causes a release of calcium ions (Ca2 ) from intracellular stores while diglyceride activates yet another enzyme, this time called protein kinase C. Through these cascade of events that have been amplified by the TRH receptors, the thyrotrophs are induced to activate TSH gene transcription, synthesizing more TSH, and then releasing them into the hypophyseal portal system in order to stimulate the thyroid gland. Other notable factors that regulate TSH synthesis and secretion are estrogen and testosterone serum concentration, other transcription factors such as Pit-1, and cAMP concentration inside the cell (Mariotti