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THE AUSTRALIAN NATIONAL UNIVERSITY

THE AUSTRALIAN NATIONAL UNIVERSITY. Introduction to Practical 3 Christian Stricker ANUMS/JCSMR - ANU Christian.Stricker@anu.edu.au http:/ /stricker.jcsmr .anu.edu. au/Practical_3.pptx. Aims of Practical 3. Overall Aim

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THE AUSTRALIAN NATIONAL UNIVERSITY

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  1. THE AUSTRALIAN NATIONAL UNIVERSITY Introduction to Practical 3Christian StrickerANUMS/JCSMR - ANUChristian.Stricker@anu.edu.auhttp://stricker.jcsmr.anu.edu.au/Practical_3.pptx

  2. Aims of Practical 3 Overall Aim • To understand the factors, which determine the generation of action potentials. Specific Learning Objectives • To measure the threshold conditions for firing an action potential. • To determine the minimum time interval between two action potentials. • To measure the relationship between the action potential discharge frequency (F) and the amplitude of the stimulus current (I). • To become familiar with the properties of the Na+ and K+ currents, which underlie the action potential. • Part 1: Action potential threshold – What is the minimum stimulus for an AP? • Part 2: Refractoriness – How soon can a second AP be elicited? • Part 3: AP frequency vs stimulus current –How fast can APs fire in trains? • Part 4: Voltage clamp –Properties of Na and K currents underlying APs?

  3. How the Parts Are Related • The parts in this practical are related: • Action potential threshold depends on activation kinetics; mostly of the Na+ channels. • Refractory periods depend mostly on Na+ channel inactivation. • Firing frequency depends on interaction between Na+/K+ channels and current stimulus. • Last part provides the experimental evidence which underpins the observations above. • It may be best to reconsider the first 3 parts after you have done and analysed part 4.

  4. Aims of Lab 3 Part 1: Action potential threshold – What is the minimum stimulus for an AP? Vary the amplitude & duration of the stimulus current step. Measure the current that “just” evokes an AP. • How does threshold ampli-tudedepend on duration? • Why? NOTE: AP threshold is often called ‘rheobase’

  5. 1. Concept of AP Threshold • Corresponds to the voltage above which the cell fires an action potential. • Question: Is this threshold fixed? • Bigger picture: excitability of cells • What are the determinants? • Mostly Na+ channel activation/-inactivation • To a lesser extent K+channel activation • The size of the conductance • AP threshold also called rheobase.

  6. Aims of Lab 3 Part 2: Refractoriness – How soon can a second AP be elicited? Vary the amplitude & delay of the 2nd stimulus current Determine the conditions for eliciting a second AP • ‘Relative’ vs ‘absolute’ refractory interval • Why this behaviour? NOTE: An AP is defined to occur when its peak is above 0 mV

  7. Concept of Refractoriness • When two APs are evoked in quick succession (< 15 ms; note squid), the 2nd AP may be absent or much smaller than 1st AP. • Consequence of Na+ channel inactivation and re-activation. • Shapes the “threshold” and amplitude of the 2nd AP. • Absolute refractory period: time during which no AP can be evoked. • Relative refractory period: time during which an AP can be evoked, but its size is smaller than 1st AP (and typically has longer half-width).

  8. Aims of Lab 3 Part 3: AP frequency vs stimulus current –How fast can APs be fired in trains? Vary amplitude of stimulus current (step of 1 s) Count number of APs elicited → frequency of APs • Plot an F-I curve • What is the shape of this curve and why? NOTE: The program automatically counts the number / frequency of APs (in notebook)

  9. Aims of Lab 3 Part 4: Voltage clamp –Properties of Na and K currents underlying APs? Impose (‘clamp’) a Vm step Measure the peak INa & IK as well as gNa and gK NormalisedgNa • Calculate peak conductance of INa & IKusing I = g (Vm – Eion) to find g -120 -80 -40 0 +40 • Measure steady-state inactivation of INa Membrane potential (mV)

  10. Concept of Voltage-Clamp • The advantage of CC is that it is simple as the membrane voltage can change. • The change is dependent on 2 factors: Cm and Rm. • If the voltage is held constant, then there is no capacitive current. • All current change is solely due to membrane current. • If we know the membrane current and the reversal potential (Erev) for the ion, we can calculate the conductance (g = I / (Vm - Erev)). • Characteristic of ionic current.

  11. Running the Simulations • If you want to run the simulations in G112 (PC), go to http://wattlecourses.anu.edu.au, then download & run the installer once. • Read the hints at the beginning of the lab notes. • Measure all amplitudes as a difference between two cursors: one on baseline, the other on peak. • Move cursor with left / right arrow keys. • Magnify areas using the mouse to drag a box around the area of interest, click in the box, then select ‘Expand’. • Revert to original axes by ‘Edit → Undo Scale Change’. • Strategy for finding thresholds, etc. • Find the right ‘ballpark’ value by making coarse guesses, then home in by making small changes.

  12. Passive Membrane Properties Action potential threshold Refractoriness

  13. Passive Membrane Properties Istim • The electrical resistance between the inside and outside of the cell via all the leak channels is ‘R’(voltage-independent) Voltage-independent ‘leak’ channels (determine Erest) Vm Istim • ‘R’ is commonly called: • RN= resistance of the neuron, or • Rin= input resistance of the neuron • If we inject a current Istim into the cell, the voltage across the membrane, Vm, will change by an amount given by Ohm’s law: ΔV = IstimRin ΔV Vm Istim

  14. Passive Membrane Properties Istim • However, the cell membrane has a capacitance(also voltage-independent) called Cm. Voltage-independent ‘leak’ channels (determine Erest) Vm Istim • Cmstores charge (remember the rubber balloon analogy) and slows down changes in Vm. • Hence, the change in Vm is slowed down at the start and end of the Istim step: Cm The change in Vm follows an exponential decay, with a time constant called the membrane time constant, tm(“tau-m”). Vm Istim

  15. Passive Membrane Properties Istim • This passive response scales up and down as Istim is made larger and smaller, but it does not change its shape. Voltage-independent ‘leak’ channels (determine Erest) Vm Istim • The passive response is due to the intrinsic properties of the membrane, and is always present when a stimulus current is applied. Cm Vm Istim

  16. Passive Membrane Properties Istim • Activation of voltage-dependent ion channels (e.g.NaV or KV channels) now adds active membrane responses. Voltage-independent ‘leak’ channels (determine Erest) Vm Istim • The active responses sit on top of the passive responses. Active response Cm Passive response Vm Istim

  17. Passive Membrane Properties Istim • The input resistance, Rin, of the cell can be calculated from the plateau value of Vm using Ohm’s law. Voltage-independent ‘leak’ channels (determine Erest) Vm Istim • However, if Istim lasts for much less time than the membrane time constant, the plateau will not be reached. Active response Cm V Passive response Rin = Istim V Vm Istim

  18. Hints for Writeup(see also Lab Handbook 2014) • Paraphrase the aims of each part of the experiment • Present primary data (tables, plots) but be selective in what primary plots you show • Plots: • Don’t simply connect the dots. • If you believe the data should fit a straight line, “fit” the line by eye or by computer and give the fit parameters (slope, intercept). • If you don’t think the data fit a straight line, you can superimpose a curve (by eye) or a ‘spline’ or ‘polynomial’ fit (by computer), but there is no need to give the fit parameters. • See the Lab Handbook for other advice about labeling axes, statistics, etc. • Answer the questions and do the things in the boxes in the lab notes • Be succinct! Reports are due Tues. 22 April, 5 p.m.

  19. Things to Remember • Operational definition for 2nd part: an AP is a potential change that reaches a peak voltage of ≥ 0 mV. • On Windows, there are two applications running: IGOR and an instance of terminal (black window). Don’t close that window as it is used to run Neuron in the background. If you Quit the simulation, close the terminal window, too.

  20. That’s it folks…

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