Transcript Nerve Excitation Slides

Nerve Excitation
Topic II-2
Biophysics
Neurons
Motor neuron
Sensory neuron
• Nerve signals due to modulations of membrane potential
• Motor Neurons have dendrites
History
 Action potential is
electrical pulse that travels
much slower than
electrical current
K+ and especially Na+
play big role
Calamari
Andrew
Fielding
Huxley
Alan
Lloyd
Hodgkin
Hodgkin and Katz: as
[Na+] decreases velocity of
action potential decreases
• Hodgkin and Huxley observe membrane potential
reverses during pulse and goes to +100 mV
• They also see that conductance of membrane increases
40X and propose have transient changes in Na and K
conductance → Nobel Prize
Voltage Clamp
Positive current:
+ ions out of
axon
VK = -72 mV,
VNa = + 55 mV
DV = Vin - Vout
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Measure current to keep voltage constant
INa = gNa(Vm – VNa), IK = gK(Vm -VK), IL = gL(Vm – Vk)
Voltage Clamp makes IC = Cm dV/dt = 0
Itot = INa + IK + IL (with clamp)
Vm = Vh = 1/gtot (gKVK + gNaVNa + gLVL + I), clamp and look
at I
Voltage Clamp experiments
•Set concentrations of ions so can get Nernst potentials
equal membrane potential and reduce variables
•gNa, gK ~ 0 at resting potential and they are only activated when
the axon is depolarized (becomes less negative). H&H get gL
when hyperpolarize
•I = IL = gL (Vm – VL), VL < Vresting (-60 mV)
Action Potential
A. Resting Na m gate closed h gate
open; n-gate closed
B. Sufficient initial depolarization →
open m-gate, Vm → VNa;
h
C. h gate closes (inactivation) and K
n-gate opens, Vm → Vk
D. n-gate closes and h-gate opens
(de-inactivation), Vm → Vrest
Do Axon 1 Dynamics
Axon 1 Dynamics
Set up is in current clamp mode. See Vm and gtotal as well as VNa and VK.
1. Run. Note how Vm goes towards VNa and then comes down and overshoots. [Can get
one firing if hit arrow next to stop/go
2. Lower VNa (this would be like reducing external sodium): 30, 0 mV. Look at effect.
Why does action potential not go as high?
3. Put VNa back to 50 mV. Increase VK to -65. What do you see?
4. For HW, explore the temperature dependence.
H&H Voltage Clamp experiments
• When depolarize, get early negative current and later positive current
•As DV increases
•Amplitude of Ineg decreases; DV = 115 have Ineg = 0; DV > 115 have early positive
current
•Rate of current development increases (both + and -)
•Switch from negative current to positive gets earlier
•Note: VK = -72 mV, VNa = +55 mV, Vresting = -60mV; INa = gNa(Vm – VNa), IK = gK(Vm – VK)
•So IK always positive; INa negative for small DV but becomes positive for DV>115 mV
•I reversal could be due to cessation of early I- or stronger/earlier I+
•H&H isolate currents by setting Vm = VNa so get voltage dependence of IK
•Deduce voltage dependence of INa since Itot = INa + IK
Do Axon 2&3 Voltage Clamp Currents
Axon 2&3
Axon 2 – Voltage Clamp and Currents
Screen shows graph with membrane potential steps and currents vs time.
Current is sum of Na and K currents. Ieak is subtracted out.
1.
Run. Start at -60 mV. Have Vstep = 10 mV.
2.
Step up Vstep by 20 mV up to 140 mV). What do you see?
3.
Find voltage when only have positive current. (~115 mV). What
is happening here?
Axon 3 – Voltage Clamp and Separate Currents
4.
Repeat Vsteps like in Axon 2. Set Vstep to 60 mV just to show
general currents. Why no sodium current at 115 mV?
5.
Go to parameters in top parameters window. See that VNa is 55
mV (so when step 115 mV = -60 + 115 = 55 mV).
6.
Now, like H&H, can change VNa (would be changing Na
concentrations) and study voltage dependence of gk alone. Take both
VNa and Vstep down by 20 (go to 35 mV and 95 mV).
7.
Could keep doing this. H&H get INa by subtracting IK from Itotal.
Na activation and inactivation and deinactivation
DV  activation , time inactivation  deinactivation
deinactivation is also voltage dependent
H&H use conditioning steps:
Brief conditioning depolarization  reduced INa during 2nd
step
As Dt for conditioning step increases  INa 2nd step
decreases (more sodium channels inactivated during
conditioning step)  H&H find time and voltage
dependence of inactivation
ex – conditioning step + 29 mV  tinactivation = 2 ms (nearly complete)
step + 8 mV  tinactivation > 8 ms (less inactivation)
They found that even at resting potential, many sodium channel
are inactive.
 H&H used long conditioning steps to study de-inactivation
This turned off (closed) Na channels then set 2nd voltage to
recovery voltage – vary time to 3rd voltage to look at current.
Do Axon 4
Voltage
Clamp Na
Inact
Axon 4 Voltage Clamp Na
inactivation
This is like Axon 2&3, but only have INa. Have two voltage steps. First, conditioning step or
pre-potential, is used for inactivation, Vpre. Second, test step, is to assess how much inactivation
there is, Vstep.
1. Vpre = 15. Dpre = 0. Vstep = 50 mV. Play (stop/Go). Get Na current.
2. So that sum of Dhold and Dpre = 15 ms, set Dpre = 1,,2,5,10 ms and change Dhold
accordingly. What is happening here?
3. Set clock to zero (click on hour glass and hit zero). Make Dhold 10 and Dpre = 5 ms.
Change Vpre to -15 and then -20 and then 25. What is happening here?
Empirical Equations
Generally dg/dt = (1-g) – g;  = opening (fwd rate),  = closing
g  g   g   g 0 e
t
t
, with g   steady state,
g 0  initial state, t  characteri stic time  1
 
Potassium, have gk = gkmaxn4; gkmax = max conductance (all open)
n  n   n   n 0 e
t
t
, with n 4  fraction of channels open
Do Axon 5
Sodium, have gNa = gNamaxm3h, m is like n for K, h describes inactivation Voltage
Clamp Na K
Each has time dependence like n
conductance
m3 = fraction activated, h is fraction de-inactivated
C m dV
dt
 g Na max m3h Vm  VNa   g K max m 4 Vm  VK   g L (Vm  VL )  I
for each of m, n, h have equation like n  n   n   n 0 e
t
t
If know parameters (max g, taus, nernst potentials etc) can get action
potential and explain all observed phenomenon.
Axon 5 Na K conductances
This is like previous exercises except, now look at conductances which you
would get from the currents divided by the potential. Remember can get g
from I = gV.
1. Play. Have 10 mV Vstep. See nothing in gNa and small effect on gK.
2. Increase step 30, 50 , 70, 115 [Vstep in middle parameter – increase].
What is happening here? Why is gNA so high so big when Vm = VNa
(Vstep = 115 mv)?
Action Potential
Do Axon 6
Impulse
conductance
Axon 6 Current clamp Summary
Hit Stop/Go
Explain what you see.
Threshold and Refractory Period
http://www.biocrawler.com/encyclopedia/Action_potential
http://cwx.prenhall.com/bookbind/pubbooks/morris5/medialib/images/F02_03.gif
There is a minimum initial depolarization you need to get action
potential – this is the threshold. “All or none” aspect of action
potential
After action potential, threshold is infinity (Na h gates closed),
this leads to a refractory period.
Do Axon 7&8
Impulse
Threshold
and
Refractory
Axon 7 Impulse Threshold
This is set up so you can find the threshold membrane depolarization for an action potential.
Hit pay twice (ignore initial). Initial current stimulation of 1 mA/cm2 is not sufficient to
do much of anything. May want (need) to hit arrow nex to stop button so only goes
once).
2. Increase current (amplitude) until see action potential (all or none).
3. Find threshold
4. Unhide gNa in lower window. Click on V on bottom. Then drag gNa to box on graph and
hit go. See small membrane response below threshold is due to transient Na activation.
1.
Axon 8 Absolute and Refractory
Periods
This exercise is like the last, but explores the effect of previous depolarizations on the threshold.
V, gk, gNa, %inAct (% inactivated Na channels), are shown.
1.
2.
3.
4.
5.
Hit play (might have to do 2x). Stimulator amplitude originally at 8 mA/cm2 which is
just above threshold. Have current impulse that gives action potentials.
Hit 2x Pulse OFF box and open parameter modification window for 2x Pulse delay. Now
2x Pulse should be on and you will have two current stimulations that can be delayed w/r
to each other.
Decrease time between pulses (echo delay), from 20 ms to 15 or 16 ms in steps of 1 ms.
See that eventually no longer get second action potential.
With 16 ms delay, show can get 2nd action potential by increasing current impulse
amplitude from 8 to 9 mA/cm2.
HW – look at cause of refractory period: channel conductances.
Spread of Action Potential
http://www.arts.uwaterloo.ca/~bfleming/psych261/image25.gif
Na comes in and causes depolarization at neighboring sites. Refractory
period insures unidirectional event.
Want current to go along axon, not out of axon
Invertebrates: large radius → small R
Vertebrates: large R along axon (myelin sheath and nodes of Ranier)
•Have ligand gated channel (eg Ach receptor that needs two Ach to
open).
•When open both Na and K can get through → get depolzarization