Electrophysiology refresher

Question 1

What is electrophysiology used for? What can it be used on? What does it measure?

Answer 1

Electrophysiology is used to measure electrical activity in biological tissue
•	Can be used on:
o	Brain
o	Spinal cord
o	Heart
o	Nerves
o	Eyes
o	Cochlea
o	Muscles
Generally, cells have negative membrane potentials – electrophysiology measures this!

Question 2

What is the primary cause of a negative membrane potential?

Answer 2

The primary cause of a negative membrane potential (Vmem) is an imbalance of K+ ions inside and outside the cell; there are more K+ inside the cell, and fewer outside.

Question 3

What are the different preparations used for electrophysiology?

Answer 3

• Dissociated neuronal cultures:
o Pros: easily accessible cells for intracellular recording
o Cons: no anatomical correlation; cells not in physiological environment; can only study early developmental stages
• Acute brain slices:
o Pros: local circuits intact; can study any developmental stage; anatomically relevant
o Cons: long range inputs/outputs severed; not in physiological environment
• Whole animal:
o Pros: all circuits intact; can correlate activity with behaviour
o Cons: technically very challenging, especially for intracellular recordings

NB: DNCs must be embryonic cells in order to be cultured; the culturing process will kill any cells harvested from adult tissue!

Question 4

Recordings can be made with which 2 pipette types? What are their differences?

Answer 4

Whole cell recordings can be made using sharp pipettes (has high tip resistance and punctures a hole in the cell), or with patch clamp pipettes (have low tip resistance, and you can perfuse the cell with the pipette solution – the cytoplasm is then continuous with the pipette interior).

Question 5

What is Ohm’s law?

Answer 5

V=IxR
Where:
V = Voltage; occurs due to separation of charge – measured in volts
I = current; represents flow of charge – measured in amps
R = resistance; opposes the flow of the current – measured in Ohms (Ω). Resistance is proportional to the number of open ion channels; more open channels = less resistance!

Question 6

What is the Nernst equation?

Answer 6

Ex = (61.5/z)log10([x]out/[x]in)
E = equilibrium of ion X
Z = ion’s valence (charge)
X = ion
NB: In adult neurons, ECl is < Vrest; ∴ adding Cl- --> hyperpolarisation. In juvenile neurons, ECl>Vrest, so adding Cl- -->depolarisation. Usually occurs BEFORE development of glutamatergic neurons!

Question 7

What is the KCC2? Why is it useful for electrophysiology?

Answer 7

The K+/Cl- co-transporter, KCC2, pumps Cl- ions out of the cell, therefore contributes to ECl
KCC2 not expressed early in development, so early in development, [Cl-]inside is higher and ECl is more depolarised.
Therefore, activating a Cl- channel early in development leads to a depolarising response.

Question 8

What is patch clamping used for?

Answer 8

Clamp keeps Vm at the desired level (X). If the channel is opened at a voltage above (e.g.) ECl, I increases. If below, I decreases.
Current moves to counteract what the cell wants to do.

Question 9

What can ephys be used to study?

Answer 9

o	Can be used to study:
	Brain
	Heart
	Spinal cord
	Nerves
	Cochlea
	Eyes
	Muscles

Question 10

What is potential difference, how does it occur, and how is it measured? What is its typical value?

Answer 10

• Neurons have a difference in charge on either side of the phospholipid bilayer – cell’s exterior is positively charged; interior of cell is (relatively) negatively charged
o Known as potential difference
• Electrophysiology measures potential difference between an electrode within the cell and an electrode outside the cell (the ground)
o Known as membrane potential
o Typically between -50 and -90mV

Question 11

What is the ionic basis of potential difference?

Answer 11

• Using K+ as an example:
o Imbalance of K+ ions inside and outside the cell (relatively more outside the cell, so outside of cell is positive)
o Anion:K+ ratio is greater outside the cell, aids relative positivity
o K+ leaky channels  buildup of negative charge inside the cell
• Membrane potential represented by: Vm=Vi-Vo
o Membrane voltage = interior voltage – exterior voltage

Question 12

How does Ephys measure the activity of ion channels?

Answer 12

• Done via carrier ions used by the channel in question – usually:
o Na+
o K+
o Cl-
o Ca2+
 Are others but these are the most important
• Neurons classified via:
o Carrier ions
o Genetics
o Activation mechanism – voltage-gated/mechano-gated
o Pharmacology
• Can measure effects of other receptor types, with electrophysiology (e.g., GPCRs)
o Can only do this via their effects on ion channels

Question 13

How can you study synapses with ephys?

Answer 13

• Usually induce AP in presynaptic neuron, then record the response activity in the postsynaptic neuron
• Current clamp ‘sets’ the membrane potential to a known value, to measure the membrane current

Question 14

What do extracellular recordings measure? Why?

Answer 14

• Can measure the change in voltage inside a cell by measuring the voltage in that neuron’s immediate extracellular space
o Used to simultaneously measure the charge change in groups of synapses
• As positive ions move INTO the cell, the extracellular space becomes more negative
o Known as a field potential, or fEPSP

Question 15

How do we study the properties of ion channels using electrophysiology?

Answer 15

• Problem:
o Want to study voltage-dependence of voltage gated ion channel
o Which membrane potential does it open at?
o Current clamp recording – membrane potential constantly changes
• Solution:
o Fix the voltage at particular membrane potentials, and study the underlying current
 ‘voltage clamping’
• In a voltage clamp, we measure the current (I) required to keep voltage (V) at a fixed level
o Analogous to a toilet water flow

• Extracellular current will be negative to maintain the membrane potential
o Must inject negative current to oppose the flow of positively-charged ions via the Glu receptor