Answer Happy

Question : Explain what is meant by (i) chronotropic agents and
(ii) inotropic agents. Give an example of a chronotropic agent.
Channel #1 Channel #2 ON OF 2 Filter Settings Gain Setting Offset Setting Input Input IN Sme H/25 OAME Output Channel 42 Output ME Channel #1 Contato Figure 1: Photograph of the front and back panels of the ETH-256, dual channel amplifier. The photos in Figures 1 show the dual channel amplifier that we will use in this lab (and others to follow). Dual channel means it can individually amplify two independent input signals. Note the paired input connectors and the fact that either channel can take either a force transducer or just measured biopotentials. When used with the force transducer, the amplifier provides the power to drive a bridge circuit for accurate readings of deflection of the transducer blade. Note also that the outputs from the amplifier are on the back of the unit, as are the power switch and power cable connector.
Oscilloscope iworx ETH 256 Bioamplifier AD Converter Force transducer Bipolar electrode Bipolar electrode Figure 3: Circuit diagram for the recording of contraction and electrograms from the frog heart. Note detail at figure bottom of the connections for bipolar electrogram.
Methods 1. Setting up the measurement circuit according to Figure 3 (force transducer component): (a) Connect the force transducer to the CH 1 8 pin DIN input of the iworx ETH-256 dual channel bioamplifier. (b) Place a T-connector on the corresponding output of the bioamplifier and then connect one end to the input (CH 1) of the oscilloscope and the other to the input (0) for the computer A/D converter using BNC cables. (c) Adjust the settings on the bioamplifier to get a clean signal on the oscilloscope in which you can see the response to gentle bending of the force transducer. Start with the following settings on the bioamplifier: Low pass filter (LPF) at lowest frequency setting (5 Hz) • High pass filter (HPF) at DC for the Frank-Starling measurements and possibly at 0.03 or 0.3 Hz for the subsequent measurements. Gain at x5-x100, adjust this as necessary. On the oscilloscope, try the following settings (make sure all settings are in calibrated mode, i.e., latched into fixed settings): • DC coupling • 200 m V-5 V/div -0.5 s/div (d) Launch the acquisition program (CB8ChScope) computers for acquiring the signals. Then select sampling parameters from the program (sampling rate of 100-200 is adequate) and run it to make sure it acquires a signal. Note: Once you have obtained a clean signal, record all settings (amplifier and acquisition program) in your lab notebook. If you ever change a setting, make a note of the new setting and when you changed it in your notebook as well.
2. Calibration of the force transducer (see Figure 4). Note that calibration can occur at the start or the end of the experiment. (What would the advantage be of one option over the other?): (a) Mount the transducer in the magnetic stand and rotate the transducer so the flat side of the blade is parallel (horizontal) to the table; deflections in the up and down direction should cause the signal on the oscilloscope to change. (b) The zero-offset is controlled via the offset knob on the bioamplifier. Adjust the offset accordingly to make maximum use of your recording range. Because we do not anticipate bi-directional movement of the transducer blade (the heart only tugs in one direction), setting the offset to zero will waste half our recording range. Therefore set the offset such that the entire voltage range displayed on CB-Scope (and the oscilloscope) is utilized. (c) Weigh a set of 3-4 paper clips of different sizes and then hang them alone and in combination from the transducer blade and note the total weight and the associated deflection of the signal on the oscilloscope (or computer). The resulting table of values will be the basis for calibration of the transducer. (d) When you are done, rotate the blade of the transducer back into the vertical orientation and, if necessary, reposition the offset. You should now be ready to perform the measurements of the contracting heart.
Methods 1. 2. Obtain a pithed frog from the lab TA/Instructor and fix the frog on its back using the big needles in the pan. Open the thorax of the frog with a central incision and two flaps. Go to the point of the Layer One section and focus on the heart. The point here is not to perform a detailed dissection but to make you familiar with the general anatomy and comfortable with the preparation of the animal. Do not cut or remove any organs other than the skin and some of the ribs covering the heart. To expose the heart, make sure to remove the lower and middle sections of the rib cage as they will interfere with the transducer you will use to measure contraction. Cut low enough so that added drugs are able to drain from the thorax. The heart of the bullfrog is quite large and red and should be slowly beating. Figure 5 shows the process of removing the ribs and sternum with the exposed heart below. If the frog is still cold, the rate may be very slow so run some Ringer's solution over the heart to help it warm up. Observe the atria and single ventricle of the heart and note the sequence of contraction of each. Once the heart is open, regularly apply a few drops of Ringer's solution to keep it moist. If you have a camera available, take photos of the thorax and mark organs on them. If you do not have a camera, find images from a classmate and label them for your lab report. 3. 4. a
La Force Transducer Bipolar Electrode Bipolar Electrode (1ಂದ Figure 6: Photos of the complete frog preparation including the thread (enhanced in orange) connecting the heart to the transducer. 5. Attaching transducer to the frog (See Figure 6): (a) Very carefully, cut open and remove the pericardium from the heart so you can see it fully exposed. (b) Using the curved needle and suture provided, run the needle through the lower part of the ventricle, about 3-5 mm from the apex of the heart, and tie a loop with the suture
thread. Then clip off the needle and discard it carefully in the sharps container (red plastic). Run the other end of the suture through the hole in the transducer blade and tie a knot there as well. Make sure there is at least 30 cm of suture available between the heart and the force transducer. (c) Place the transducer at the end of the pan, elevated 5-10 cm above the edge of the dissection pan with the blade oriented perpendicular to the thread. The thread from the frog heart to the transducer should be quite flat (horizontal) so that you apply tension to the long axis of the heart. See Figure 6 for reference. (d) Use alligator clips to attach a wire between the metal dissection tray and the large metal plate on which you are working. This can reduce the electrical noise levels substantially when we start to perform electrocardiographic measurements. (e) Now apply enough tension to the thread such that you see a signal on the oscilloscope that reflects the contraction of the heart. Sensitivity of the oscilloscope should be in the range of 500 mV-5V/div, depending on the gain setting of the amplifier. Adjust location and tension so as to generate as clean a signal as possible, ideally one that reveals the separate components of atrial and ventricular contractions. Make sure the tension of the thread is just enough to pull the thread taught and lift the heart slightly. Check also that there is no obstruction from the side of the pan or any other object. Place the pan and the stand well away from the edge of the lab bench and always be careful not to touch the post or the thread accidently. Otherwise, any change in orientation will alter the resting tension and the reference signals, which will add error to subsequent measurements.
2.5 Acquiring electrograms Heart Bipolar Electrode Figure 7: Exposed heart with applied bipolar electrodes. The electrodes should touch the exposed heart lightly on the surface of the ventricles.
Methods Now, to visualize and acquire the “electrogram”, the bioelectric potential from the surface of the frog heart, carry out the following steps referring to Figures 3 and 7: 1. Obtain three BNC to banana converters/splitters. Connect the BNC outputs from the electrode to two of the BNC/banana adapters. Connect the two black banana ends together. Connect the two red banana ends to the remaining BNC/banana adapter and connect it directly to the input of the bioamplifier. To further improve signal quality, use alligator clips to connect the common negative poles to the ground on the frog (pan and plate) and to the oscilloscope ground (use a banana cable). 2. Attach the bipolar electrode to the second input channel of the iworx ETH-256 bioamplifier. 3. Attach a T-connector to the second output of the bioamplifier and split the output between the second channel of the oscilloscope and channel 1 of the A/D converter. 4. Try the following settings on the bioamplifier: High pass filter (HPF) at 0.03 or 0.3 Hz. The purpose of this filter is to remove the low frequency drift. Low pass filter (LPF) at 50 or 150 Hz. The purpose of this filter is to limit high frequency noise; too low a setting also removes information at cardiac frequencies, especially important for the electrical signal. Gain typically in the range of x10 to x100 but adjust as needed to get good quality signal. 5. Attach the bipolar electrode to a magnetic stand that can lift up and down, and place the electrodes in contact with the heart surface. It should just lightly touch the ventricles but experiment with electrode placement to achieve strong signals. For additional improvement in signal and to reduce sensitivity to heart motion, try placing the electrode underneath the heart, i.e., so that the heart sits on top of the electrode. .