NMJ application manual
Note: In this tutorial only that part of the program that concerns electrical stimulation of the muscle will be used.
Run the Action potential program. A dialog box appears reading "Please enter you name". Type in your name and press Enter. Your name will appear inside the top row of a table located in the lower left corner of the screen.
The program uses a model experiment. Let us familiarize ourselves with the overall design of the experiment first. Select Help/Preparation item of the main menu. The following description of the experiment will be displayed:
The preparation consists of a small piece of diaphragm with phrenic nerve, isolated from an anesthetized laboratory rat. The preparation is placed in a Petri dish and bathed with a Krebs solution containing 95% of O2 and 5 % of CO2 at a temperature of 32 °C. The Krebs solution can be modified by the addition of drugs and its ionic composition can be changed as well. The Petri dish is placed under a dissecting microscope. A glass microelectrode with the tip diameter of less than 0.1 µm is inserted into the motor end plate. The muscle fiber can be activated either trans-synaptically by stimulating the nerve (we will ignore this option now), or by direct injection of the depolarizing current.
After familiarizing yourself with the experimental setup, click on Continue. The experimental setup will be replaced with a standard oscilloscope screen on the left and clipboard viewer on the right. The oscilloscope will display the membrane potential of the muscle fiber as detected by the glass microelectrode. The time base of the oscilloscope is set to 16 ms and is switched on 1 ms before the actual stimulation.
Muscle stimulation: "Stimulate" (menu item)
"'Muscle" (submenu item)
Use numeric keyboard to change both the amplitude and duration of the stimulation.
After pressing the Stimulate rectangle, an oscilloscope animation of the membrane potential will begin. The first millisecond is reserved for the resting membrane potential. The rest of the trace displays a change of the potential depending on the values of our parameters (amplitude and duration). The sweep ends after 16 ms.
Storing the trace in clipboard:
"Clipboard" (menu item)
"Copy to Clipboard" (submenu item)
The clipboard can be erased by clicking on "Clear Clipboard.
You will use the clipboard to store and compare the action potentials generated by different experimental treatments of the preparation.
Changing the concentration of ions: "Ions"
Select a specific ion in submenu, enter its concentration and click OK. The current value of concentration will be displayed in a small box located in the lower left corner of the screen. In order to return to default physiological concentrations of all ions, press "Reset to normal" (last submenu item in "Ions").
Drug administration:
"Drugs" (menu item)
A table called Add Drug will appear. Select a drug of your choice and enter the desired concentration. In this tutorial, we will use tetrodotoxin (TTX) and 3,4-diaminopyridine. The concentration of the drug will be shown in a small box in lower left corner of the screen below the ion names. After the experiment with drug administration do not forget to wash the preparation with default solution by clicking on "Clear All Drugs".
Evaluation of the action potential changes:
- Resting membrane potential
- Amplitude
- Speed of depolarization and speed of repolarization
Experimental tasks
Trial 1
Determine the threshold value of depolarization current after evoking a typical action potential with a stimulus lasting 10 ms. How will the action potential change when the stimulus amplitude grows by 2 nA?
Trial 2
Use 10 nA for stimulation and apply it for 0.5 ms in the first case and for 10 ms in the second case. Explain the changes of action potential that you observe. Comment on the phenomenon that occurs when the stimulation is very long.
Trial 3
Use 10 nA current lasting 1 ms and modify potassium concentration between 3 and 10 mM/l. Describe changes of the resting membrane potential that you will observe. Note the shape of the action potential during the stimulation. Explain the relationship between the extracellular concentration of potassium and threshold for stimulation.
Describe the effect of 1 mM/1 of the potassium channel blocker 3,4-diaminopyridine that you will add to the solution. Explain its effect on action potential.
Trial 4
Describe and explain the effect of tetrodotoxin (sodium channel blocker). How little of TTX is still effective? Can the effect of TTX be compensated by an increase of extracellular concentration of sodium? What does that mean?
