Action Potentials in Earthworms
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Experiment AN-4: Action Potentials in Earthworms Background In the resting cell, the permeability of the membrane to potassium (P K) is greater than its permeability to sodium (P Na ). Stimulation, like synaptic activity coming from other nerve cells, can depolarize (make less negative) the cell membrane. Sodium channels in the cell membrane are sensitive to membrane depolarization and they respond by opening, which increases the membrane’s permeability to sodium. If the depolarization reaches or exceeds a certain level (threshold), an action potential is produced. Action potentials develop because of a regenerative, positive feedback cycle. As the cell’s permeability to sodium increases, sodium conductance increases, and increased sodium conductance leads to greater depolarization of the membrane. As depolarization increases, sodium permeability increases again, and more voltage-sensitive channels open. With more channels open, sodium conductance and membrane depolarization increase until the membrane potential reaches the equilibrium potential for sodium. But, before the equilibrium potential for sodium is reached, two other events occur: the voltage- sensitive sodium channels close soon after they open, and the voltage-sensitive potassium channels open. With these channels open, potassium ions leave the cell and cause the membrane to repolarize (hyperpolarize) towards its resting level. This process of membrane hyperpolarization closes the voltage-sensitive potassium channels and re-primes the sodium channels so that they are ready to open once more. This is called the refractory period. Propagation of the action potential from the site of initiation to other locations along the nerve cell is caused by the positive charges in the cell leaking to an adjacent (unstimulated) region and depolarizing that region enough to create an action potential there. In this way, the signal moves from one region of the axon to the adjacent one, and ultimately to the end of the axon. Some axons are myelinated; the axon is covered with a series of Schwann cells, a type of glial cell which electrically insulates the axon. The spaces between adjacent Schwann cells are called the nodes of Ranvier, and they are the only regions along the axon where the membrane is exposed to the extracellular fluid. The myelin insulation prevents the currents associated with action potentials from leaking out of the membrane until they reach a node. So, action potentials take place only at the nodes in myelinated cells. In this laboratory you will record action potentials from the ventral nerve cord of an annelid, the earthworm Lumbricus terrestris. The ventral nerve cord of some invertebrates is a structure analogous to the dorsal nerve cord of vertebrates. The ventral nerve cord of an earthworm contains three giant neurons. The cord has one medial giant neuron with a lateral giant neuron on either side of the medial neuron. Because of their size, the three giant neurons in the earthworm nerve cord can generate action potentials with conduction velocities that permit the worms to have a fast escape reflex. When a large stimulus is delivered to the nerve cord, the neurons respond and action potentials from the medial and lateral neurons are seen. You will examine certain principles associated with neuronal activity: • Neuron viability—determining the viability of the neurons in the nerve cord by observing action potentials from the medial and lateral giant neurons. • Thresholds of neurons—determining the stimulus amplitudes needed to generate action potentials from the medial neurons. Animal Nerve – Earthworm Action Potentials – SetupIXTA AN-4-1 • Conduction velocity—measuring the speed at which an action potential propagates down the medial neuron. • Effects of temperature—observing how cooling affects the conduction velocity of the medial neuron. • Stimulus strength and duration—observing how the stimulus amplitude needed to generate an action potential is related to the duration of the stimulus pulse. Animal Nerve – Earthworm Action Potentials – SetupIXTA AN-4-2 Experiment AN-4: Action Potentials in Earthworms Equipment Required PC or Mac Computer IXTA data acquisition unit USB cable IXTA power supply iWire-B3G input cable NBC-402 Nerve Bath Chamber C-BNC-P2 Stimulator Cable - BNC to Dual pin jack stimulating cable C-ISO-FP5 Recording Cable - 5 Lead Pin jack recording cable Room-Temp & Chilled Ringer's solution Bottles containing 10% ethanol solution IXTA Setup 1. Place the IXTA on the bench, close to the computer. 2. Check Figure T-1-1 in the Tutorial chapter for the location of the USB port and the power socket on the IXTA. 3. Check Figure T-1-2 in the Tutorial chapter for a picture of the IXTA power supply. 4. Use the USB cable to connect the computer to the USB port on the rear panel of the IXTA. 5. Plug the power supply for the IXTA into the electrical outlet. Insert the plug on the end of the power supply cable into the labeled socket on the rear of the IXTA. Use the power switch to turn on the unit. Confirm that the red power light is on. Start the Software 1. Click on the LabScribe shortcut on the computer’s desktop to open the program. If a shortcut is not available, click on the Windows Start menu, move the cursor to All Programs and then to the listing for iWorx. Select LabScribe from the iWorx submenu. The LabScribe Main window will appear as the program opens. 2. On the Main window, pull down the Settings menu and select Load Group. 3. Locate the folder that contains the settings group, IPLMv4Complete.iwxgrp. Select this group and click Open. 4. Pull down the Settings menu again. Select the Action Potential-Worm-LS2 settings file from Animal Nerve. 5. After a short time, LabScribe will appear on the computer screen as configured by the Action Potential-Worm-LS2 settings. Animal Nerve – Earthworm Action Potentials – SetupIXTA AN-4-3 6. For your information, the settings used to configure the recording channels in the LabScribe software and IXTA for this experiment are are programmed on the Channel window of the Preferences Dialog. 7. The settings used to configure the stimulator for this experiment are listed in Table AN-4-S1 . Both groups of settings are programmed on the Channel and Stimulator windows of the Preferences Dialog, which can be viewed by selecting Preferences from the Edit menu on the LabScribe Main window. 8. Once the settings file has been loaded, click the Experiment button on the toolbar to open any of the following documents: • Appendix • Background • Labs • Setup (opens automatically) Table AN-4-S1: Settings on the Stimulator Window of the Preferences Dialog that Configure the iWorx System for Experiment AN-4. Parameter Units/Title Setting Stimulus Mode Pulse Stimulator Start With Recording Time Resolution msec 0.01 Toolbar Step Frequency Hz 1 Toolbar Step Amplitude Volts 0.01 Toolbar Step Time Sec 0.0001 Delay Sec 0.005 Amplitude (Amp) Volt 0.200 Pulses (#pulses) Number 3 Pulse Width (W) msec 0.1 Time Off (T Off) msec 10 Time Off Amplitude Volts 0 Holding Potential (HP) Volts 0 Animal Nerve – Earthworm Action Potentials – SetupIXTA AN-4-4 Earthworm Recording Setup 1. Plug the BNC adapter of the C-BNC-P2 stimulator cable into the stimulator 1 input of the IXTA stimulator ( Figure AN-4-S2 ). The banana plug that goes into the negative (black) stimulator output is identified by a tab, embossed with the letters GND (ground), on that side of the adapter. 2. Locate the C-ISO-B3G recording cable ( Figure AN-4-S1 ) in the iWorx kit and plug it into the iWire 1input on the front of the IXTA ( Figure AN-4-S2 ). Figure AN-4-S1: The C-ISO-B3G recording cable. Figure AN-4-S2: The C-ISO-B3G recording cable and C-BNC-P2 stimulator cables attached to the IXTA. Animal Nerve – Earthworm Action Potentials – SetupIXTA AN-4-5 Figure AN-4-S3: NBC-402 Nerve bath chamber with the C-BNC-P2 and C-ISO-FP5 cables for recording worm action potentials. Electrical Noise Electrical noise is the most common problem associated with the recording of bioelectric signals. It radiates through the air and comes from electrical devices in the lab room or building: lights, power outlets, computers, monitors, and the power supplies. Since the source of power for these devices is 60Hz alternating current (AC), this electrical noise appears as a distorted sine wave with a repeating period of 16.7 milliseconds (msec). There are two major sources of electrical noise: pickup and ground loops. Pickup Pickup is caused by electrical radiation that produces currents in the electrodes and wires leading to the amplifiers in the recording system. Because the resistance in the electrodes is high, small currents produce large voltages that may be greater than the biopotential being recorded. The major ways to reduce pickup are: • Faraday Cage: Put a grounded, screened enclosure, known as a Faraday cage, around the preparation and the electrodes. the enclosure separates the source of the radiation from the electrodes. The person operating the equipment might also be a source of noise, and he or she may need to be grounded. • Shielded Cables: Use shielded cables to carry the signals from the electrodes to the amplifier and the recorder; this puts a protective ground around the wires carrying the bioelectric signal. • Differential Recording: Record using both a positive and a negative recording electrode placed on a nerve or neuron. The noise signals that are equal in magnitude, but opposite in polarity, will cancel each other out and leave a flat baseline. • Short Cables: Use the shortest cables available to reduce the length of wiring exposed to electrical noise. Animal Nerve – Earthworm Action Potentials – SetupIXTA AN-4-6 • Direct Current Equipment: Use equipment, like preamplifiers and illuminators, that are powered by batteries or direct current (DC) transformers.