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Voltage Clamp Protocol
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Procedure

 

Neuron Simulator is an online simulating platform for a section of excitable neuronal membrane of squid axon. Simulator uses Hodgkin and Huxley equation and provides full accesses to HH parameters, membrane parameters, various ionic concentrations, pharmacological effects, stimulus parameters and Vclamp protocol.  Loading Neuron simulator by clicking on the tab called “simulator” (see the below sample screen shot (Fig.1.) of the simulator).

 

Fig 1. Screen shot of the Neuron Simulator

 

 The simulator consists of two main sections: simulator window and variables menu. The simulator window shows the membrane potential plot and plots of various parameters. The upper plot shows the membrane potential vs. time plots (red), stimuli just below the Vm(blue) and lower plot displays a variety of parameters including HH-parameters, various currents and conductance any three variables at a time can be plotted in this window, by default I_leak,Ina,IK was plotted.
 


Click on the Stim1/Stim2 buttons to inject depolarization / hyperpolarization current to neuronal membrane. Simulation parameters can be modified in Membrane window, Channels window, Drugs window, Stimuli windows (see the figure (Fig.2) below).

 

Fig 2. Variables menu

 

Simulation control window



Simulation control menu is used to simulate the experiments. The functionalities includes, Run button (will continuously simulate the neuronal membrane, without giving any input stimuli), Stop button (will help to stop the stimuli at any point of time), Stim1 and Stim2 buttons (are two are used to inject an external stimulus as some nA of current to the neuronal membrane, user can modify this by clicking Stim1 and 2 window). Simulation runs for around 30ms when a stimulus (Stim1/Stim2) is applied, Export button (this functionality of this simulator will give user to the freedom of accessing/exporting the simulated models data to an excel sheet, by using these data user can make high resolution pictures) and finally the Reset button (helps to reset the whole simulator).

Fig 3 . Simulation control menu

 

Membrane window


Membrane window provides accesses to internal and external ionic concentration and membrane properties.
 

Fig 4 . Membrane tab

 

Channel window



Channel window provides accesses to various parameters of passive as well as active channel types. Individual channel can be disabled by making the conductance of that channel to zero.

Passive channels conductance can be varied in the channel window. Voltage gated channels parameters can be accessed by clicking on channel details. Channel details will display a new detailed window with channel properties of fast sodium, delayed rectifier, user defined channels. The figure given below shows the detailed channel properties of fast sodium channel.

 

Fig 5. Channel tab

 

Stimuli window

 


Stimuli window consists of two external stimuli (Stim1 and Stim2) which can be set by user, each of which consists of either single pulse or a sequence of two independent adjustable pulses.

Fig 6 . Stimuli tab

 

Drug window



Drug window allows to study the pharmacological effect in neuronal membrane by the application of three drugs TTX (Inhibit Na current), TEX (Inhibit K current), Pronase (eliminate Na+ inactivation).At any point of time these drugs can be applied.


 

Fig 7 . Drugs tab

 

Procedure for Voltage clamp experiments



By default the simulator will be in current clamp mode, for every reset the simulator the mode will reset to current clamp. Voltage clamp protocol tab of the simulator can be accessed by clicking the drop down list in the variables menu and selecting Voltage clamp mode as shown in the below fig 8. Note that the stimuli tab changed to voltage clamp stimuli.



 


Fig 8. How to select voltage clamp mode


In this exercise we will study the contribution of Na+ and K+ currents (because of in flow and out of these ions) as major contribution to total current  generated by a nerve cell. Effect of drugs like TTX and TEA on these channels as pharmacological perspective. This exercise will also help to understand how the parameters recorded from an voltage clamp experiment were used to formulate the kinetics of voltage activated ion channels responsible for excitation (Na+, K+, fast gates, slow gates, etc.)
 
 

 

 


Fig 9. Over view of the simulator after selecting the mode as voltage clamp

 




 
Fig 10. Setting input voltage as stimuli in mV

 


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


This will simulate HH neuron model for applied voltage.


Total ionic current and individual components of ionic current is estimating and total ionic current plotting in the top graph as red trace in response to the voltage applied as voltage which is plotted as blue trace in the same graph (see fig. 11). 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 selecting the check boxes below this graph. 



 


Fig. 11  Shows the total current plotted in the top graph and its components (INa and IK) separately plotted in the bottom graph. 

 


Pharmacological studies using TTX and TEA


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. 12  Effect of TTX (tetrodotoxin) on sodium current


Tetraethylammonium is abbreviated as TEA. 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. 13 Effect of TEA (Tetra Ethyl Ammonium) on potassium current

 

 

 

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Note: Reset the simulator for every experiment
Neuron Simulator is based on David S. Touretzky's Hodgkin-Huxley Simulator.

 

Cite this Simulator:

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