1. Choose Tutorial "The Na Action Potential"

2. Increase the time base ( Total # ( ms ) ) to 20 ms

3. Open a 2nd Stimulus Control Panel

4. Set the delay on the first panel to 0 ms.

   • leave duration and amplitude set to default

5. Set the delay on the second panel to 4 ms.

   • leave duration and amplitude set to default

6. Click on Reset & Run

7. Repeat this stimulation protocol with increases of the 2nd delay , in increments of ms

1. Describe what you observed , and propose a possible mechanism

   • When the second current injection is delayed by 10 milliseconds , it begins to decay non exponentially ,

     • indicating asdf

   • When the delay for IClamp[1] = 11 ms ,

     • another action potential can be triggered again

   

8. Repeat this stimulation protocol for 2nd delays of 4 , 5 , 6 , 8 , and 10 ms

   • increasing the 2nd stimulus amplitude to determine whether you can generate a 2nd action potential.

   • try doubling the amplitude repeatedly

2. Indicate what stimulus amplitude produced the most “action potential” like response.

   2nd Delay ( milli second )	Necessary Amplitude for AP ( nano Ampere )
   4	3.2
   5	1.6
   6	0.8
   8	0.4
   10	0.2133

3. Indicate what happened to threshold and action potential peak as the 2nd delay was increased.

   $$\text{2nd Stimulus Delay Time}\frac{1}{\propto }\text{Threshold}$$
   $$\text{2nd Stimulus Delay Time}\propto \text{Action Potential Peak}$$

4. Explain what could account for these changes in threshold and peak

   • the delay has to be at least long enough for sodium channels to inactivate and then reset to closed

   • the cell also has to wait on potassium channels to close and reset.

   

9. Set the 2nd stimulus delay to 10 ms

   • with an amplitude sufficient to generate an action potential

10. Select Keep Lines

11. Click on Reset & Run.

12. Now change the 1st stimulus to an amplitude of 0.5 nA and a duration of 8 ms.

5. Indicate what happened to the action potential triggered by the 2nd stimulus

   • it blocked it from happening

   

6. Indicate whether a 2nd action potential occurred after increasing the 2nd stimulus amplitude.

   • if you increase to around $0.9nA$$0.9\ nA$ , then you first start to see deflection in repolarization phase of action potential

   

7. Explain what events could have caused this to happen

   • most of the sodium channels are still inactivated

   • we got it to fire again because the first set potassium channels that opened during first action potential were still not closed

13. Close 2nd Stimulus Control Panel.

14. Set 1st stimulus amplitude to 0.0 nA

15. Set Total # ( ms ) to 30 ms

16. Increase Extracellular K ( mM ) in 1 mM increments

8. Explain what accounts for these electrical responses

   • $[K^{+}{]}_{\text{extracellular}}=7mM$$[\ K^+\ ]_{\text{extracellular}} = 7\ mM$ :

     • first action potential fires

   • $[K^{+}{]}_{\text{extracellular}}=8mM$$[\ K^+\ ]_{\text{extracellular}} = 8\ mM$ :

     • second action potential fires

   • $V_m\propto E_{K^{+}}\propto [K^{+}{]}_{\text{extracellular}}$$V_m \propto\ E_{K^+} \propto [\ K^+\ ]_{\text{extracellular}}$

   • the more you increase extracellular potassium , the more positive $V_m$$V_m$ becomes

     • this is generally going to move $V_m$$V_m$ closer to the threshold ,

       • making it easier to fire action potentials