Frog Heart In this experiment, you will explore basic principles of cardiac muscle physiology by recording force of contraction and the electrocardiogram (ECG). You will also examine the effects of temperature, stretch, and the neurotransmitters acetylcholine (Ach) and norepinephrine (NE) on heart function. ______________________________________________________________________________ Background Studies of isolated organs were pioneered in the late 19th century when scientists such as Sidney Ringer (1835-1910) developed perfusion solutions designed to replicate extracellular fluid such as plasma (e.g. Ringer’s solution). These physiological salt solutions (PSS) could sustain isolated organs for extended periods. By adding washed red blood cells (RBCs) to Ringer’s Solution, a more physiological perfusate could be produced. Addition of RBCs meant greater oxygen carrying capacity for the solution. A classic example of an isolated organ preparation is the frog heart. After euthanasia of a frog, its heart will continue to beat in situ for several minutes (hours if properly supported). During this time, basic cardiac functions can be investigated. Historic examples of such experiments are those performed at University College London by Ernest Starling (1866-1921, middle image), his brother-in-law, William Bayliss (1860-1924, left image), and Otto Loewi (1873-1961, right image). Collectively, the experiments of these men and their collaborators led to the coining of terms such as ‘hormone’ (Starling and Bayliss), identification of the first hormone, ‘secretin’ (Bayliss and Starling), introduction of the fields of ‘endocrinology’ (Starling and Bayliss) and ‘neurotransmitters’ (Loewi), developed the Frank Starling Law of the Heart (Starling), and identified acetylcholine (Ach) as the first known neurotransmitter (Loewi). Loewi then shared the Nobel Prize in Physiology_1936_ with Sir Henry Dale whom he met while doing experiments in the laboratory of Ernest Starling. The heart is composed of cardiac muscle. Cardiac muscle is similar to skeletal muscle. Both are striated. Among the differences in the two muscle types, cardiac muscle exhibits refractory periods (meaning that a second stimulus given shortly after a first stimulus will not elicit a response) that are not seen in skeletal FROG HEART muscle. Cardiac muscle also displays automaticity and spontaneous rhythmicity that are not seen in skeletal muscle. Most cardiac muscle cells have the innate ability to depolarize and contract spontaneously (skeletal muscle does not). Cycles of spontaneous depolarization and repolarization occur rhythmically, giving rise to an intrinsic, regular heartbeat. In the intact, normal mammalian heart, heart rate is determined by the sinoatrial node (SA node or cardiac pacemaker). Spontaneous depolarization, in the absence of external influences, is called: automaticity, diastolic depolarization, and/or pacemaker potentials. In the frog heart, the sinus venosus is the functional equivalent of the mammalian SA node (Figure 1). Figure 1. Ventral and dorsal views of the frog heart. Oxygenated blood from the frog’s lungs returns to the left atrium whereas deoxygenated blood returns to the right atrium. The single ventricle receives blood from both atria so there is some mixing unlike in mammals and pumps it out through one large artery, the truncus arteriosus. Despite what has just been written, the structural organization of the frog ventricle, and its inflow/outflow tracts, is such that there is minimal mixing of the two sources of blood. The consequence is oxygenated blood is delivered to the brain and most tissues, whereas deoxygenated blood flows to the lungs and skin (Figure 2). Besides the lungs, frogs can exchange gases (and water) through their skin. Figure 2. Cross-section of frog heart 2 FROG HEART In addition to cell-to-cell current flow, there is a specialized conduction pathway (Figure 3). This provides the only electrical connection between the atria and ventricles – the AV node. Although there are pacemaker cells here, conduction is much slower than it is through either atrial or ventricular muscle. This allows the depolarization of the atria, and thereby atrial contraction, to be completed before the ventricles depolarize and contract. Mammals have two ventricles while the frog has only one. From the AV node, the action potential travels at 1 m/s into the Bundle of His and left and right bundle branches to the Purkinje network. Purkinje fibers penetrate the entire ventricular mass, from apex to base. Conduction in this network, 5 m/s, allows the ventricles to behave as a syncytium. Figure 3. Electrical conduction system of mammalian heart While no external stimulii are required to initiate/maintain the heartbeat, the heart receives continuous input from the sympathetic and parasympathetic nervous systems. Cardiac muscle responds to a variety of neurotransmitters and other agents that can increase or decrease heart rate and contractility. These molecules are able to change the rate of spontaneous depolarization of the pacemaker cells, for example, in the sinus venosus. At rest, the vagus nerve inhibits the sinus venosus, so that the resting heart rate is slower than the intrinsic rate. Stimulating the vagus nerve electrically can slow the heart (bradycardia) and even cause it to stop (vagus-induced cardiac arrest). In such a situation, Ach will eventually be hydrolyzed by cholinesterase enzymes and contractility will be reinitiated as part of the phenomenon called ‘vagal escape’. Stimulating sympathetic nerves causes tachycardia or increased heart rate. The strength of cardiac contraction is influenced by the degree of stretch of the ventricular muscle. This relationship is referred to as the Frank/Starling law of the heart. The name comes from the nearsimultaneous discovery by Otto Frank of Germany and Ernest Starling of Great Britian of these principles. Both are given credit for the discovery. Their Law states that the strength of contraction (atrial or ventricular) is influenced by the degree of stretch of cardiac muscle. Up to some maximum, the greater the stretch at the end of diastole, the greater the strength of the subsequent systolic contraction, and therefore the stroke volume (Vs). Increased venous return to the left ventricle increases left ventricular end-diastolic pressure (LVEDP) and volume, thereby increasing ventricular preload, or degree of atrial filling and stretch. The normal operating point is at a LVEDP of approximately 4-8 mmHg and a Vs of approximately 70 ml/beat (in humans). This provides an intrinsic mechanism by which cardiac output can match venous return and also be maintained if aortic pressure rises. This relationship is affected by circulating norepinephrine which increases the strength of contraction at any degree of stretch (Figure 4). 3 FROG HEART Figure 4. Frank/Starling Law normal curve. Frogs are poikilotherms, which means they do not internally control their body temperature. Instead, a frog’s body temperature is determined by the temperature of its environment. As a general rule of thumb, increasing temperature increases metabolic functions, and decreasing temperature decreases metabolic functions (e.g. elevated heart rate, decreased heart rate). In this experiment we will look at two temperature manipulations using warm and cold Ringer’s solutions. Their effects on the heart will be compared with room temperature conditions (normothermia). Required Equipment o o o o o o o o o o o o o o LabChart 8 software PowerLab 26T Data Acquisition Unit with a Bio Amp Force Transducer Small weight (5 grams) Ring Stand Micromanipulator and clamps 3-5 Lead Shielded Bio Amp Cable Shielded Lead Wires (3 Alligator Clips) Strong thread Barbless hook Syringes One medium-sized, euthanized, bullfrog Instruments/tools: o Glass Petri dish o Sharp scissors o Blunt probe o surgical tray with wax or pad o surgical pins Solutions: o Frog Ringer’s – room temperature o Frog Ringer’s – cold (in a 5-10 oC water bath) o Frog Ringer’s – warm (in a 55-60 oC water bath) o Acetylcholine (0.1 mM) o Norepinephrine (1 mM) 4 FROG HEART Procedure Calibrating the force transducer Raw output from the Force Transducer, like all other ADInstruments physiological transducers used in this lab, is expressed in millivolts (mV). The transducer needs to be calibrated to give more meaningful physiological units of force such as grams, (e.g. 5-10 g). Physiological transducers also have residual voltage that has to be corrected. Other sources of electricity in the laboratory produce 60 Hz interfering noise (e.g. overhead lights, hoods, computers, other electronic equipment). Hence the need to ‘warm up experimental equipment’ before the experiment, and to regularly ‘zero’ transducers during the experiment. 1. Open the LabChart settings file for 4 Frog Heart 2. Under “Force” dropdown menu, select Digital Filter and change Cut-off frequency to 15 Hz. 3. Under “force” dropdown menu, select 500g Force transducer. Leave the Force Transducer undisturbed. Observe the moving signal in the popup window (Figure 4) Figure 4. Bridge pod dialog box. 4. Record for five seconds, and then hang a known weight (e.g. 5 grams, provided by your instructor) from the Force Transducer. After 5 seconds click pause, then click “Units…” (see Figure 4 above). 5. Select a short segment of the red line where no weight was added, and click the arrow next to “Point 1.” Add zero (0) to the open field. 6. Select a short segment where the weight was added,and click the arrow next to “Point 2.” Add the value of the known weight in grams to the corresponding open field. 7. In the Units field scroll down to ‘Define units’ and select g. 8. Remove the weight, then click the OK button In situ frog heart preparation 1. Obtain a double-pithed frog from your instructor. Secure the animal ventral side up in a clay-filled surgical tray using straight pins. 2. Make a short midline incision at the inferior margin of the coelom, followed by bilateral, oblique incisions starting at the midline incision and cutting in a superior direction towards the forelimbs (see your TA for clarity of instructions). 3. Cuts should pass through the wall of the body cavity (incise skin and muscle wall simultaneously; be careful to not cut internal organs). 5 FROG HEART 4. Bring the bilateral incisions together near the throat and completely remove the incised, ventral flap of coelomic tissue. 5. Carefully grasp the pericardium with fine forceps, snip with scissors and gently remove the pericardium to expose the heart (Figure 5). Figure 5. Heart exposed. Figure 6. Pierce heart at apex. 6. You will be provided with a 15-centimeter length of suture with spring-loaded hook clamps fixed to each end. Attach one hook near the apex of the ventricle. With considerable slack in the line, attach the other hook to the MLTF500 force transducer (see Figures 6 and 7). 7. Raise the force transducer on the ring stand until there is no slack in the thread and it is near a 90 angle from the bench surface in all perspectives. Adjust the tension (micromanipulator) so it is taut. 8. Attach the Lead Wires (with Alligator Clips) to either the pins holding the frog or to heart tissue (preferable). Attach the Channel 1 positive lead (red +) to the left forelimb or near/on the left atrium. Attach the channel 1 negative lead (red -) to the right forelimb (near/on right atrium), and earth lead (black) to the left hindlimb (or to tissue beneath the ventricle) (Figures 8 and 9). Make adjustments until you get the cleanest, reproducible waveforms on the computer monitor. 6 FROG HEART Figure 7. In situ heart preparation. Try attaching leads near heart. Exercise 1: Recording Baseline Data In this exercise, you will examine baseline heart rate and contractility observed from the force generated during repetitive cardiac cycles. Note: It is important that you watch what is happening to the heart and what is being recorded on the monitor. You might be asked to describe your observations in the Data Notebook (or on a quiz). 1. Zero the force transducer by opening the “500g Force transducer” pop-up and clicking “Zero”. You do not need to recalibrate the force transducer. 2. Start recording. Add ‘baseline observations’ as a comment. Observe the signal. If you have a weak signal in the Force channel, increase the tension on the heart with the Micromanipulator but be careful not to over-tighten the thread. If your ECG signal is poor, check the connections to the frog. Also make sure the frog is not too close to the computer monitor. The computer can cause electrical interference. 3. Record good data for one minute. Stop recording, but do not close the file. Note: You want to keep the frog heart moist throughout the experiment. Use the wash bottle of RT (room temperature) Ringer’s solution after each exercise to superfuse the heart. Analysis Exercise 1: Recording Baseline Data 1. Examine your data in the Chart View (top tool bar icons). Autoscale, if necessary. 2. Use the View Buttons to set the horizontal compression so you can see all the baseline data at once. 3. Select the baseline data from the Heart Rate [Force] channel. Make sure you only select the data trace and include all peaks and valleys in your trace. Click the second icon in the Data Pad Toolbar “Add to Data Pad”. Note: At the end of the recording, no data are present. If part of the trace is selected without any data, the Data Pad will not display the mean heart rate (Figure 8). 7 FROG HEART Figure 8. Proper data selection for the heart rate [Force] channel. 4. Open the Data Pad by clicking the first icon in the Data Pad section of the toolbar. 5. Record the mean heart rate in your lab notebook (Table 1) as shown in the Data Pad of this document. 6. Look for the Heart Rate [Force] Maximum and Heart Rate [Force] Minimum columns in the Data Pad. Record the maximum and minimum values (Table 1). 7. Repeat steps 3-6 for the baseline data from the ECG channel. 8. Look at the values from these two channels. How do they compare? Record your observations in the data notebook. Exercise 2: Effects of Temperature In this exercise, you will examine the effects of temperature on the heart. You will compare cold and warm Ringer solutions. 1. Zero the force transducer as before. 2. Measure the temperature of the wash bottle of RT Ringer’s solution (at room temperature, e.g. 24 oC). Record the value in Table 2 of the Data Notebook. 3. Using the same file, Start recording. Add a comment with “exercise 2.” Record baseline data for 30 seconds. Do not stop recording until the end of the exercise. 4. Measure the temperature of the wash bottle of warm Ringer’s solution (e.g. 55-60 oC). Record the value in Table 2 of the Data Notebook. 5. Superfuse the heart with 3-5 mL warm Ringer’s solution. Add a comment with “warm.” Record for 46 minutes. 6. Measure the temperature of the wash bottle of cold Ringer’s solution (e.g. 0-10 oC). Record the value in Table 2 of the Data Notebook. 7. Superfuse the heart with 3-5 mL cold Ringer’s solution. Add a comment with “cold.” Record for 4-6 minutes. 8. Stop recording. Superfuse the heart with room temperature (RT) Ringer’s solution. Wait several minutes before moving on to the next exercise. Analysis Exercise 2: Effects of Temperature 1. Examine the data in the Chart View, and Autoscale, if necessary. 2. Select 5-10 cm of data from the heart rate [force] channel when heart rate is rhythmic and monitored variables are in/near their steady states (for room temp, hot and cold solutions). 8 FROG HEART 3. Click the second icon in the Data Pad section of the toolbar (Add to Data Pad). Click the first icon to open the Data Pad and view the data that was added. 4. Record the mean heart rate in your Table 2 from the Heart Rate [ECG] channel (column D). 5. Repeat steps 1-4 for the hot and cold Ringer’s solutions. Exercise 3: Frank/Starling Law of the Heart In this exercise, you will investigate the Frank/Starling Law of the heart: Up to some maximum, the greater the stretch at the end of diastole, the greater the strength of the subsequent systolic contraction. 1. Zero the force transducer as before. Using the same file, Start recording. Add a comment with “exercise 3.” 2. Record 10 seconds of baseline data. 3. Continue recording. Slowly increase the tension on the heart by 1.0 mm by turning the micromanipulator knob. Add a comment with “stretch 1.0 mm.” Continue recording until contractility is in a new steady state. 4. Repeat step 3 for a minimum of three 1.0 mm stretches (up to 5, depending on viability of heart). 5. Immediately return the micromanipulator to its original position to reduce tension on the heart. 6. Stop recording, but do not close the file. 7. Superfuse the heart with RT Ringer’s solution. Wait several minutes before moving to the next exercise. Analysis Exercise 3: Frank/Starling Law of the Heart 1. Examine the data in Chart View, and Autoscale, if necessary. 2. Select baseline data in the Force channel. You can use the Zoom View, but it is not required. 3. Place the Marker on the baseline data trace just prior to the first beat. Use the Waveform Cursor to determine the amplitude of the first beat. 4. Repeat steps 1-3 for each stretch condition. Do not move the Marker. Include the amount of stretch in Table 3 of the Data Notebook. 5. Record each of the values in your lab notebook (Table 3). 6. Use the ECG channel to determine the mean heart rate for each stretch condition, using the Data Pad as in Exercise 1. Record these values in Table 3 of the Data Notebook. Exercise 4: Effects of Neurotransmitters In this exercise, you will examine the effects of two neurotransmitters, acetylcholine (Ach) and norepinephrine (NE), on the heart. Acetylcholine is released by postganglionic parasympathetic nerves onto the heart, whereas NE is released at the heart by sympathetic postganglionic axons (also by the adrenal medulla). Note: Be sure to apply the neurotransmitters in this exercise in the order indicated. Between each step, allow the heart to recover for several minutes and superfuse with fresh Ringer’s solution. 1. Zero the force transducer as before. 2. Using the same file, Start recording. Add a comment with “exercise 4.” Record 30 seconds of baseline data. 3. Using a 1.0 ml tuberculin syringe (with 26g needle), inject 0.1 ml of NE (1 mM) directly into the ventricle. Add a comment with “norepinephrine.” Record for several minutes (e.g. 4-6), then Stop recording. 9 FROG HEART 4. Superfuse the heart with Ringer’s solution, and allow it to recover for several minutes. 5. Record 30 seconds of data after superfusion with Ringer’s solution. 6. Superfuse the heart with 1 ml Ach (0.1 mM). Depending on the viability of the heart (determined, in part, by time since extraction and quality of preparation and waveforms recorded), Ach might cause cardiac arrest. It should ‘escape’ from this condition after a few minutes. If it does not see your TA for further advice (e.g. readminister an intracardiac bolus of NE). Analysis Exercise 4: Effects of Neurotransmitters 1. For each neurotransmitter determine the HR from the ECG channel as in Exercise 1. 2. Record these values in your lab notebook (Table 4). 3. Calculate the percent change in heart rate for each neurotransmitter using the following equation: 4. Record these values in your lab notebook (Table 4). Data Notebook Table 1. Comparison of resting heart rate Calculated from Force Channel Calculated from ECG Channel Mean Heart Rate (bpm) Maximum Heart Rate (bpm) Minimum Heart Rate (bpm) Table 2. Effects of temperature on heart rate Mean Heart Rate (bpm) Normal Ringer’s (___) oC Warm Ringer’s (___) oC Cold Ringer’s (___) oC 10 FROG HEART Table 3. Effect of tension on force and rate Heart Contractile Force (g) Mean Heart Rate (bpm) Baseline (No Stretch) Stretch 1 (___) mm Stretch 2 (___) mm Stretch 3 (___) mm Stretch 4 (___) mm Stretch 5 (___) mm Table 4. Effects of neurotransmitters on heart rate Heart Rate Before Drug Administered (bpm) Heart Rate After Drug Administered (bpm) Norepinephrine Acetylcholine 11 Percent Change in Heart Rate How to write a laboratory report for Systems Physiology Laboratory (01:146:357) Abstract (≤ 350 words) In short, the abstract provides a summary of the experiment you conducted. Readers generally read the abstract to get a feel for the topics that the paper will address. Therefore, the abstract must cover the main points of your experiment. The abstract has five parts that must be addressed for every experiment: 1. An introductory statement 2. The purpose of the study 3. Methods that were used to obtain data 4. Results from each main experiment 5. Significance of your findings (discussion of mechanism) The introductory statement broadly introduces the main topic to the reader. This section can include an eye-catching statement, encompassing themes, or more specific background information related to the research topic. The purpose of the study is to provide the reader with the rationale for conducting the experiments and must be as specific as possible. In order to write an effective purpose, you must understand WHY you performed the experiment. Ask yourself the following questions: Was the experiment conducted to examine a physiological concept in more detail? Was it to observe the behavior of a tissue/organ in response to a physiological stress? It may also include any primary measures you took to reach your purpose. The abstract must also include a brief methodology. As many of you are aware, various methods can be used to test one hypothesis. Including your methodology in the abstract allows the reader to visualize the way you obtained your data. Be brief and keep in mind that specific information regarding the methods used in the experiment can be found in the methods section. Important information to include would be the subject population (e.g., mice, humans), the medium tested (e.g., blood, urine, muscle), and the manner that it was tested (e.g., glucometers were used to measure blood glucose concentrations). It is also important to mention any information related to equipment setup and if the experiment required repeated testing at specific intervals. The results section of the abstract is to briefly list the main results you obtained. Ask yourself if you saw specific trends or values, which will depend on the experiment being conducted. Be sure to list the results for ALL experiments that you have conducted and will be discussed in the full lab report. The last part of your abstract should include a brief statement about the significance of your findings. What did you learn from the results? Did they support conventional theories? Did they deviate from established findings? When doing this, describe what the conventional theories or established findings are. Describe the mechanism that led to your results. As you continue to read through the guidelines for preparing a laboratory report (i.e., how to write an introduction, methods, results, discussion, and reference sections) refer and use the sections of the journal article(s) posted on Canvas to reinforce your understanding of each section’s content. Introduction (1-2 pages) There are two reasons for writing an introduction: 1. to inform the reader of the basic scientific concepts behind the experiment 2. to state the purpose and hypothesis of the experiment If the reader has a basic proficiency in the scientific concepts studied in the experiment, he/she will be more appreciative of the results and will understand the significance of the findings. To write an effective introduction, imagine explaining the physiological concepts to someone with a basic background in biology. Remember, start gradually and broadly. Describe the anatomy of the system you are addressing in your report. If the experiment requires that the tissue/organ be placed under physiological stress conditions, describe how the tissue/organ would behave under normal conditions. Do not delve into details, as these will only confuse the reader. Importantly, avoid discussion material. Pretend you are writing the introduction to an experiment without already knowing the results. The information provided in the introduction section should eventually lead to a clear and concise purpose statement. Following a purpose statement for each aim, a hypothesis should be included. Again, the hypothesis should be based on information already provided in the introductory material. References should also be used in the introduction to provide support for background information and hypotheses. Methods (1-2 pages) The purpose of the methods section is to summarize all the steps you took as a researcher to conduct the experiment. The methods should not omit important details and should not include superfluous points. The methods should be formatted to include subsection headers such as Participant, Equipment, Procedure, and Data Analysis. DO NOT include the following: ● Unnecessary details, such as, “The frog’s skin was green and the toes had a black mark on them.” This has no relevance to the experiment. ● A numbered/bulleted list. The methods section should be written in paragraph form. ● Pronouns or personal information of participants/subjects. ● Copied sentences from course material, this is considered plagiarism. Organize your methods so that it parallels the experiment. Start with a brief description of the subject(s) and then mention any dissection protocols or equipment preparation. Then proceed to describing each experiment. Include important information relative to each experiment (e.g., doses, time intervals, etc.) and the way you analyzed your data. For the data analysis subsection you should inform the reader of the outcomes you measured and the methods you used to obtain your data. Did you use any specific equations? Did you draw any figures? Be brief but mention these important points. Do not include any results in your methods section. Results (1-3 pages) For the purposes of this lab course, graphs and tables will be used to depict trends in data. The results section is organized in the following manner: ● The results summary describes the data collected in each experiment. This section reports the overall message of the data and addresses more specific details of your data. Are you showing an increase? Decrease? Maintenance of values? You should adequately detail the data you are presenting (i.e. reporting data at different time points and/or between groups). The summary can be limited to a few sentences for each experiment. You can include the summary at the beginning of this section or embed the text between your required figures and tables. ● A pictorial representation of data, this can either be a graph or table. ● The caption describes the figure or table. This information is right beneath the corresponding graph or table. A caption should include a general statement about the data presented. This section also explains aspects of the figure/table such as defining units and reporting sample size. Resources for graphing data: • Calculating mean and standard deviation: https://www.youtube.com/watch?v=39JDN5BNEJ0 • Adding custom standard deviation bars to figures: https://www.ablebits.com/office-addins-blog/add-error-bars-excel/ Discussion (1-3 pages) This section of the report carries the most weight as here you are explaining the reason behind the patterns/trends in your data. Organize your discussion section so it flows to the reader. If your discussion section is not organized, your readers will not follow your explanations even if they are sound. In this section of your report, include a body paragraph for each of the main experiments that you are required to report results on. This section can be organized according to each variable studied, or grouped to explain a theme in the data. The latter can be used when the experiment calls for investigating different aspects of one principle (e.g., muscle activity can be studied by changing the length of the muscle or the frequency). In each body paragraph, remind the reader of your aim(s) and provide an overview of your results. For the purposes of this course, you should address physiological principles when explaining your data. If your data deviate from expected trends, explain the expected trend and why this would occur, then provide an alternative explanation for your data. Following the body paragraphs, you should describe any limitations or sources of error in the experiment within one paragraph. The discussion should end with a conclusion paragraph. Within a few sentences, provide a summary of the study and results to reader. Your conclusion should also acknowledge the overall significance of the experiment and your findings. References (1 page – not included in page count) DO NOT forget to provide citations in your report. These are vital in providing the reader with proof of your claims. Facts discovered by someone other than you have to be credited to the right person. Not providing a citation indicates that this is a fact you are stating based on your own conclusions. Neglecting to give credit where credit is due constitutes plagiarism, which is a serious offense! Bottom line; provide in-text citations throughout your report. Plagiarism – One must do his/her own work when writing reports in System Physiology Laboratory. It is permissible to use short excerpts/quotes from the work of others; however, in any single report, these should be few and short. In all cases where the work of others is cited, credit must be given. When citing, avoid using the same exact words as an author or paraphrasing large segments of writing. Read the information and explain it in your own words. Do not cite Canvas material, lecture notes, manuals, or TAs. Please use the format given to you by your TA when citing textbooks and journal articles in your reports! For instance, if your TA asks you to use APA formatting, be sure to locate a credible source that can guide you with the citation process. Below is a link that provides useful information on APA formatting:https://owl.english.purdue.edu/owl/ resource/560/07/
Purchase answer to see full attachment