Introduction to electrophysiology Flashcards
What is a resting membrane potential?
The voltage difference across the membrane of a neuron when at rest
-70 mVs intracellularly
What is the concentration gradient?
The concentration of positive or negative ions higher in one area than another
What is depolarisation?
Change in a neuron’s membrane potential that makes it more positive
What is hyperpolarisation?
Change in a neuron’s membrane potential that makes it more negative
What are ionotropic receptors?
Transmembrane proteins that form a channel allowing ions to travel in or out of a cell
- ligand-gated channel opens when receptor binds a ligand (e.g. NT)
Glutamate receptors and GABA-A receptors are ionotropic receptors
What are voltage-gated ion channels?
Transmembrane proteins that form ion channels whose opening and closing is regulated by the membrane potential near the channel
What is an action potential?
Process by which a neuron sends information down its axon
What are the 4 types of biological electrical activity?
- large voltages generated by animals
- e.g. electric eels or rays: < 700V - Negative resting membrane potential
- most neurons: -70mV - Postsynaptic potentials
- small variable changes in membrane potential: 1-40mV - Action potentials
- large, fast, all or none: < 100mV
What are electroplaques?
Cells in animals that generate large voltages
- e.g. electric eels or rays
How are electroplaques activated?
By NT ion channel receptors:
When fish want to give electric shock, electroplaques are activated by a nerve which releases Ach onto nicotinic-type Ach receptors: ligand-gated receptors
-> flow of Na+ into electroplaques -> depolarises -> brief potential change of 120mV
What are the gradients corresponding to the force of diffusion and the electrostatic force?
- Force of diffusion = concentration gradient
- Electrostatic force = electrical gradient
What happens to Na+ ions at resting membrane potential (Vmrest)?
Concentration and electrical gradients direct Na+ intracellularly
What happens to K+ ions at resting membrane potential (Vmrest)?
Concentration gradient directs K+ extracellularly despite inward electrical gradient
What happens to Cl- ions at resting membrane potential (Vmrest)?
Concentration gradient moves Cl- intracellularly despite outward electrical gradient
What happens to Ca2+ ions at resting membrane potential (Vmrest)?
Concentration and electrical gradients direct Ca2+ intracellularly
How are Excitatory postsynaptic potentials (EPSPs) generated?
By activation of ion channels that depolarise neurons
- Glu NT opens ion channel permeant to positive ions = inflow of Na+ and Ca2+
How are Inhibitory post synaptic potentials (IPSPs) generated?
By activation of ion channels that hyperpolarize neurons
- GABA NT opens ion channel permeant to negative ions = inflow of Cl-
Why are EPSPs and IPSPs graded in amplitude?
Due to the concentration of NT and length of time the NT is in the synaptic cleft
What are the stages of an action potential?
- Resting Vm
- Na+ and K+ channels closed, resting - Upstroke
- Na+ channels open: Na+ goes in -> depolarisation
- K+ channels closed, resting - Peak
- Na+ channels closed, inactivating
- K+ channels opening - Downstroke
- Na+ channels closed, inactivated
- K+ channels open: K+ moves out of cell -> hyperpolarisation - Resting Vm
- Na+ and K+ channels closed, resting
How does an action potential go down a neurons’ axon?
Depolarisation opens Na+ channels -> Na+ ions intracellularly -> further depolarisation -> further Na+ channels opening
What is a field potential?
The electric potential in the extracellular space around neurons
What is a nerve?
A bundle of axons
What is the compound axon potential?
The sum of the activity in a number of nerve fibres (axons)
- can be recorded extracellularly
What are the 5 versions of extracellular recording (ER)?
- Field potentials
- Whole nerve recordings
- Muticellular (multi-unit) recordings
- Single unit recordings
- Multi-electrode arrays (MEAs)
What is common to all versions of extracellular recording (ER)?
> The electrode is outside but close to the neurons
> The electrodes pick up only field potentials and low frequency filtered APs
> We cannot record Vmrest or postsynaptic potentials
What constitutes the technique of field potentials?
- Stimulating electrode
- e.g. in hippocampus: activates Schaffer collaterals - NT released onto Purkinje neurons in CA1 - Recording electrodes pick up:
- the field excitatory postsynaptic potential (fEPSP)
- the somatic population spike AP (e.g. of CA1 cells in hippocampus)
=> We record the changes associated with changing membrane potentials of neurons (EPSP and resulting AP)
What is the field excitatory postsynaptic potential (fEPSP)?
Effect of glutamate receptors opening, causing depolarisation
What is the somatic population spike action potential?
Sum of many APs
What constitutes the technique of whole nerve activity?
> Nerve isolated and placed on platinum wires
- outter chambers sealed with silicone grease, filled with olive oil to electrically isolate the nerve
- > only the AP travelling down the axons (nerve) are recorded
> Stimulating electrodes increase the compound AP amplitude
- the stronger the stimulation, the more axons are recruited to fire an AP
- > Compound AP gets larger and eventually all axons will be recruited = maximal compound AP
> Can identify subgroups of axons by conduction velocity and stimulus threshold
In the technique of whole nerve activity, how can different axons recorded be separated?
By the intensity of the stimulus and their conduction velocity
What constitutes the technique of multi-unit activity recording?
> Multi-unit extracellular recordings can be be done in vivo
> Electrode, e.g. placed in anaesthetised rat brain
> Stimulation (e.g. flash of light) to activate neurons
> Simultaneous recording of different neurons (e.g. one very close to electrode, the other further)
What constitutes the technique of single unit activity recording?
> Stimulations to characterise single axon or neuron
> Comparison of the responses from the same single unit
What constitutes the technique of multi-electrode arrays (MEAs)?
MEA electrodes are bedded in the bottom of a dish
- 64 electrodes
- inert to cells
- pick up extracellular network electrical activity
> Neurons are grown inside these dishes, in an incubator
- grow on the multi-electrode arrays
- > monitoring of electrical activity (APs) in a non-invasive manner
> Characterises single axon or neuron
What are the disadvantages of the field potentials technique?
> Cannot identify individual cells
> Cannot monitor PSPs or Vm
What are the disadvantages of the whole nerve technique?
> Cannot identify individual cells
> Cannot monitor PSPs or Vm
What is the disadvantage of the multi-unit technique?
Cannot monitor PSPs or Vm
What are the disadvantages of the single unit technique?
> Difficult to do in vivo
> Cannot monitor PSPs or Vm
What are the disadvantages of the technique of multi-electrode arrays (MEAs)?
> Cannot target particular cells
Only in vitro
Cannot monitor PSPs or Vm
What is the common disadvantage of extracellular recording techniques?
Cannot monitor PSPs or Vm
With intracellular recordings, what can the nature of the recording be?
> Current clamp
or
Voltage clamp
With intracellular recordings, what can the nature of the recording electrodes be?
> Sharp electrodes
or
Patch clamp electrodes
How are sharp electrodes used for intracellular recordings?
They penetrate the plasma membrane of the oocytes (animal’s egg)
How are sharp electrodes used in a current clamp recording setup?
We change the current and record the voltage (AP)
What is a current?
The rate at which an electric charge is flowing
How are sharp electrodes used in a voltage clamp recording setup?
We stimulate with voltage and record the current
- voltage induces the current that generates the AP
- we record the current flow through the membrane that generates the voltage pulse (AP)
How does the patch clamp technique work?
Patch clamp goes on the cell’s surface and interacts with the membrane
- forms a gigaseal on cell’s surface
What are the 5 variants of the patch-clamp technique?
- On-cell patch
- Inside-out patch
- Whole-cell clamp
- Outside-out patch
- Perforated patch
What characterises the on-cell patch technique?
Cell-attached patch recording:
- we record single ion channel activity
What characterises the inside-out patch technique?
Membrane patch is off the cell
- access to intracellular side of the patch
-> we can apply drugs and record single channel activity
What characterises the whole-cell clamp technique?
We break through the membrane with patch attached to the whole cell
- access to intracellular space -> record all ion channels in the cell’s membrane
> Whole cell patch clamp recording electrodes can be used in current-clamp and voltage-clamps mode
What characterises the outside-out patch technique?
We pull off a small patch of membrane with 1 or 2 ion channels in it
-> outside of the membrane is exposed to extracellular space (normal for the cell)
What characterises the perforated patch technique?
There is a pore forming antibiotic in electrode
- we make a gigaseal on cell’s membrane, wait for the antibiotic to diffuse to the membrane
- > membrane forms pores in itself
> Contents of cell are dialysed (separated) by electrode solution, BUT it still allows for electrical activity to be recorded
With the perforated patch technique, why do you not loose the cell’s contents?
The whole on the cell’s membrane made by the antibiotic (from electrode) are small enough
How can the whole cell patch clamp recording electrodes be used in voltage-clamp mode?
Acid stimulus:
- pH change evokes acid-sensing ion channels (ASICs) that generate current when open
- increasing acidity of the compounds increases the current’s size
How can the whole cell patch clamp recording electrodes be used in current-clamp mode?
Intracellular calcium measurement
- Ca2+ entry during AP is related to number of APs activated in that cell
-> simultaneous recording intracellular Ca2+ levels and neuron’s APs
How does the single channel recording technique work?
You can record a single channel conductance and measure its voltage dependance with voltage change in the channels
e.g.:
> Vp -10: cell depolarised by 10mV -> single channel conductance quite low, channel closed most of time
> Vp -20: one or both channels open ; increased activity ; channel current larger
> Vp -30: depolarised cell ; channel opened more ; more current
What are the 4 consecutive modes of the in vivo patch clamp technique?
- Search mode
- “strike”
- Cell attached mode
- Whole cell mode
What characterises the search mode of the in vivo patch clamp technique?
Positive pressure applied on the electrode
- forces air into electrode - pushes out fluid to keep path clear
-> the electrode can be moved through the tissue
What characterises the “strike” mode of the in vivo patch clamp technique?
Electrode hits the cell -> pulse at heartbeat frequency
- we go back to a neutral pressure on the electrode
What characterises the cell attached mode of the in vivo patch clamp technique?
Electrode giga-ohm seal is formed on cell’s membrane
What characterises the whole cell mode of the in vivo patch clamp technique?
We’re sucking the solution up - suction on electrode
-> destroys the membrane patch made under the seal = whole cell mode
=> recording of sophisticated responses in vivo
Which electrophysiology techniques can be applied on humans in vivo?
Only extracellular recording, as part of treatment
Which electrophysiology techniques can be applied on non-humans in vivo?
- Extracellular recording (implanted / anaesthetic)
- Intracellular recording (anaesthetic)
- Single cell recording (anaesthetic)
Which electrophysiology techniques can be applied in vitro (including use of human tissue)?
Extracellular, intracellular, and single cell recording
What is the disadvantage of the current clamp technique?
Cannot record voltage
What is the disadvantage of the voltage clamp technique?
Unstable
What are the advantages of the sharp electrode technique?
- Reusable
- Simple electrode solution
What are the disadvantages of the sharp electrode technique?
- High resistance
- Can be difficult to make
- Some damage to cell
What are the advantages of the patch electrode technique?
- Low resistance
- Relatively easy to make
- Less damage to cell
What are the disadvantages of the patch electrode technique?
- Not reusable
- Complex electrode solution
What are the advantages of the single channel technique?
- Allows recording in real time of the functional activity of a single protein
- Elucidates drug action at molecular level
What is the disadvantage of the single channel technique?
Complex and lengthy analysis
What are the advantages of the electrophysiological approach?
Electrophysiology is:
- dynamic
- functional
- based on international system of units
- real-time
- high-fidelity and high temporal resolution
It can be used simultaneously or in conjunction with optical, molecular, biochemical and pharmacological approaches
=> essential to the understanding of the nervous system
