L11- Advanced electrophysiological techniques

Question 1

What electrophysiological recording are done?

Answer 1

Potential difference between a rcoridng and ground electrode is measure and displayed on an oscilloscope.

Extracellular recording- measures surrounding flow of charge into and out of neurones during APs

Intacellular recording- allows measurement of the potential difference between inside and outside of the neurone.

Question 2

Which techniques use extracellular recording?

Answer 2

ERG- electroretinograms
EEG- electroencephalogram
ECG-electrocardogram
EMG- electromyogram

Question 3

What are MEAs?

Answer 3

Multi/micro electrode arrays- flat plates with multiple extracellular electrodes/channels on. Great for cell culture.
Allows continuous recordings of cells.
Allows recordings of drug responses

Question 4

What is the disadvantage of extracellular recording?

Answer 4

No indication of individual channel activities

Question 5

What did early intracellular recordings in squid axons measure?

Answer 5

Ion conductance across the cell membrane increases dramaticaly during an action potential.

Question 6

What recordings did Alan Hodgkin and Bernard Katz take?

Answer 6

Intracellular recordings
If you lower Na+ in the extracellular fluid, the amplitude of the AP is decreased.
Therefore Na+ is responsible for the rising phase of the AP.

Question 7

Why is it hard to measure the Na+ and K+ conductance at isolated membrane potentials?

Answer 7

Because the sodium and potassium channels are coupled to membrane potential. Depolarizaion causes Na channels to open, Na influx, more depolarization.

Question 8

What is the voltage clamp technique?

Answer 8

• Holds the cell at a predetermined voltage.
• Membrane potential set, depolarisation set
• Voltage gated channels open at the voltage they normally would
• ionic current in/out of the cell doesn’t change the membrane potential from the set value.

Question 9

What did Hodgin and Huxleys’ voltage clamp show?

Answer 9

Isolated elements of the AP and described the ionic steps for the first time.
Showed small capiticance and leakage currents
Showed toxins blocking particular channels.

Question 10

What do patch clamp recording of single channels show?

Answer 10

Ion fluxes/currents through single channels.

• on or off responses
• channel lifetime
• channel currents

Question 11

Who developed patch clamping?

Answer 11

Erwin Neher and Bert Sakmann- nobel prize 1991

Question 12

What is a patch clamp?

Answer 12

A glass tube pulled to giv a very fine tipped electrode- less than 1um wide

Question 13

How is the patch clamp set up?

Answer 13

• Pipette (electrode) is filled with electrical conducting solution- pipette solution.
• pippete is then connected to very fast amplifier and recording equipment.

Question 14

What is the cell-attached configuration patch clamp used for?

Answer 14

To record currents through a limited number (1-2) channels at the cell surface
Good for looking at single channel currents in response to regulation of channel by cell.

Question 15

What’s the inside-out configuration used for?

Answer 15

To record currents through a single active channel away from the cell.
Good for looking at agents which modulate channel by working at it’s intracellular face.

Question 16

What is the outside-out configuration used for?

Answer 16

To record currents through a single active channel away from the cell. Good for looking at agents that modulate channels by working at its extracellular face.
Rip off part of the membrane with the channel, hold in the air, membrane flips over, stick it in the bath.

Question 17

What is the whole-cell configuration used for? (most widely used patch clamp)

Answer 17

To record currents through active channels in the whole cell.
Good for looking at cell curretns in response to drugs added from outside or regulation of channels by whole cell.

Question 18

In the patch clam technique what happens when the channel opens?

Answer 18

Ions flow so there’s a “blip” of current.

Graph goes up then down again after.

Question 19

WHat does activation of a channel do?

Answer 19

Increases the probability of the channel opening.
No stimulus- rarely opens, spontaneously
stimulus- opens frequently

Question 20

WHat is ohm’s law? what could it tell us about the channel?

Answer 20

Current= volts x conductance

If we hold voltage constant, could determine the conductance of the channel. Tell us about selectivity etc.