Procedure

 

Click here for general introduction to the simulator.

In this exercise we are studying the relationship between ion channel properties and the V-I Curve. And which are the important parameters recorded from a voltage clamp experiment to plot V-I curve to formulate the kinetics of voltage activated ion channels responsible for excitation (Na+, K+, fast gates, slow gates, etc.).

Step 1 : Load simulator by clicking on the icon "Simulator" on top panel and select "Voltage clamp" from the drop down menu under "Variables" tab.

 
Fig 1.  Shows how to change simulation mode to "Voltage Clamp"

Step 2 : Click on "Voltage Clamp Stimuli" tab under Variables sub window and then click on " voltage clamp protocol" button to get the user friendly flexible interface to set command, test and return voltage.

 

 
Fig 2. Shows VStim user interface

Step 3 : Basically the student has to click on the brown spot which is on the Vstim (see Fig 3) plot to adjust the delay and amplitude of command, test and return voltages.

Fig 3. Shows voltage clamp stimuli in mV.

Step 4 : Press Vstim button of simulation control tab to give stimuli as voltage to the neuron as shown in the figure below.

 
Fig 4. Run control

This will simulate HH neuron model for applied voltage.

Step 5 : Total ionic current and individual components of ionic current is simulated and total ionic current plotted in the top graph as red trace in response to the applied  voltage which is plotted as blue trace in the same graph (see fig. 5). Components of ionic current (sodium and potassium currents) are plotted in the bottom graph. Other variables including  hh parameters can be plotted in the same window by enabling the check boxes below.
 

Fig. 5 Shows the total current plotted at the top plot and its components (INa and IK) separately plotted in the bottom plot. Find the peak current by moving the mouse over the plot.

Repeated the same procedure (step 1-5) by  changing the step voltage -60mV to +40mV with an increment of 10 mV at step 3 in order to complete voltage clamp protocol . In each case the peak potassium and sodium current has to be recorded. Then finally the V-I curve for sodium / potassium is plotting by assigning peak evoked INa/IK on y-axis and applied step voltage on x-axis (see Fig. 7 ).  

 
Fig 6. measuring peak Na current

Peak sodium current can be estimated directly from simulator by moving mouse over the peak of the trace (see Fig 6) or from exported file (see Fig 7).
   

 
Fig 7. Measuring peak Na+ current from exported file

Procedure for plotting Voltage Vs INa graph using Matlab / GNU Octave

Make sure that Matlab is installed in your machine (find matlab installation folder in windows C:Program FilesMATLAB , For Linux users type 'matlab /octave' on terminal)

1. Double click on Matlab icon on the desktop (for linux type matlab / octave on terminal), this will load matlab / octave in your computer.
2. Create a new matlab script file from File-> New -> Script / Open a text editor.
3. Matlab / Octave uses '%' for commenting a line, use '%' to give appropriate title for your script.
4. Assign input step voltage to a row vector called ' voltages '.
for example " voltages = -60:10:60;"
5. Assign spike frequency to a row vector called 'INa'.
for example "INa=[0,-0.0616,-0.304,-0.6413,-0.8817,-1.06162,-1.0325,-0.949,-0.7868,-0.565,-0.2993,0,0];"
6.  And finally plot the F Vs I graph (number of spikes on y-axis and injected current on x-axis). Use star symbol '*' for highlighting the data points.
for example "plot(voltages, INa,'*');"
7. Add label for x and y axis.
for example  " xlabel('Voltage mV'); "
              " ylabel('Current nA'); "
8. Save the script as 'V-vs-INa_plot.m', then run the script by clicking run button at top panel of the matlab file editor or press 'F5' button of your key board, for octave type the file name on the octave terminal from the same directory where the file is saved.
9. Matlab / Octave may ask you to change the path for execution , please click on change path.
10. The plot that you are seeing at your monitor is V vs INa plot

Procedure for plotting Voltage Vs INa graph using Microsoft Excel / Open Office

Make sure that Microsoft Office is installed in your machine (find Microsoft excel installation folder in windows C:Program FilesMicrosoft Office , For Linux users Open Office)

1. Double click on Microsoft excel icon on the desktop or Start-> All Programs -> Microsoft office -> Microsoft excel , this will load Microsoft excel in your computer.
2. Enter step voltage and peak INa as table format (like Table 1 of theory ) to the excel sheet.
3. Select the table values of both the Colum (excluding label).
4. Go to Insert-> Charts -> Line -> 2D line (select the format you wanted to plot).
5. The plot that you are seeing at your monitor is V vs INa plot (the plot will look like the figure shown below).

Fig. 6 shows the V-I relation between peaks evoked sodium / potassium current and step potential  (plotted using Matlab).
Selectively blocking ion channels to estimate individual channel's V-I curve.

Tetrodotoxin abbreviated as TTX. Tetrodotoxin blocks action potentials in nerves by binding to the voltage-gated, fast sodium channels in nerve cell membranes, essentially preventing any affected nerve cells from firing by blocking the channels used in the process. The effect of TTX drug can be simulated in Hodgkin and Huxley neuron by altering the conductance of sodium channels.

 
 
 
Fig. 7 Sodium current is diminished by TTX (tetrodotoxin)

Tetraethyl ammonium is abbreviated as TTX. It is a potassium ion channel blocker.TTX is used in neurophysiological experiments to study potassium channels. The effect of TEA drug can be simulated in Hodgkin and Huxley neuron by altering the conductance of potassium channels.
 
 

Fig. 8 Potassium current is diminished by TEA (Tetra Ethyl Ammonium)

 

Neuron Simulator is based on David S. Touretzky's Hodgkin-Huxley Simulator.
 

 

 

Help:

 

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