Electrophysiology

Question 1

What are ions?

Answer 1

Ions are charged particles.

Question 2

What does the flow of ions represent?

Answer 2

Current.

Question 3

What can electrophysiology measure?

Answer 3

Events on μs timescale.

Question 4

What are the advantages of electrophysiology?

Answer 4

• Can measure events on a us time scale
• Can measure activity of single ion channel
• Spatial resolution can be good = can tell exactly where ion channels are located
• Can tell us how channels work

Question 5

What are the main types of recording in electrophysiology?

Answer 5

Extracellular
- electrode placed outside but very close to the cell
- easy
- not very specific = gives info about the activity of a group of cells instead of a single cell

Intracellular
- electrode played inside membrane of the cell
- only useful for fairly large cells (50 um or bigger)
- hard
- specific

Patch
- used for very small cells
- very hard
- very specific

Question 6

What is the purpose of EEG?

Answer 6

Type of extracellular recording

Often done after sleep deprivation to trigger epileptiform activity.

Question 7

What are the two main methods of stimulation and recording in electrophysiology?

Answer 7

Stimulate and record.

Question 8

Who developed the patch clamp technique?

Answer 8

Sakmann and Neher.

Question 9

What are the four (five) different configurations of the patch clamp?

Answer 9

• Cell attached
• Outside-out
• Inside-out
  = allows recording of single channels
• Whole cell
• Perforated patch
  = allows recording of many channels

Question 10

What is the primary use of the patch clamp?

Answer 10

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

Question 11

What does the whole cell patch clamp configuration allow?

Answer 11

Good for looking at cell currents in response to drugs.

Question 12

What is a characteristic of a simple channel’s activation mechanism?

Answer 12

It can be either closed or open.

Question 13

What do rate constants k+1 and k-1 determine?

Answer 13

• Stability of open state
• Stability of closed state

Question 14

What can studying the patterns of openings and closings tell us?

Answer 14

About the activation mechanism.

Question 15

What does the conductance of a channel tell us?

Answer 15

About the channel pore and its functionality = amount of ions passing through the membrane

Question 16

How does current relate to ions in a channel?

Answer 16

Current is ions per second.

Question 17

What does a high conductance channel indicate?

Answer 17

Higher ion flow.

Question 18

What is the significance of looking at single channel currents?

Answer 18

They add up in the context of many channels.

Question 19

Fill in the blank: Electrophysiology allows measurement of activity on a _______ timescale.

Answer 19

μs

Question 20

True or False: Patch clamp methods can only measure a single ion channel at a time.

Answer 20

False.

Question 21

What is electrophysiology used for?

Answer 21

Measure current between a sample & surrounding medium

Can be used on organs, tissues and cells

Question 22

Mechanism of cell-attached recording

Answer 22

Starting point for all other types

Gentle suction applied to an isolated channel

Question 23

Mechanism for whole-cell recording

Answer 23

Electrode is connected to the inside of the cell
High suction disrupts the membrane that’s under the electrode

Question 24

Mechanism for inside-out recording

Answer 24

Membrane’s inner face is facing the bath solution
Begins with cell attached recording to an isolated channel
Membrane is pulled away from the cell using suction
Electrode is removed from the bath to encourage ends of the membrane to seal onto the electrode
Electrode is placed back into the bath

Question 25

Mechanism for outside-out recording

Answer 25

Outer face of the membrane is facing the bath Begins with cell attached recording Then with whole cell recording Electrode is kept in the bath so that the ends of the membrane can seal onto each other Outer face now faces the bath Begins

Question 26

Applications of inside-out cell recordings

Answer 26

Records currents through a singe active channel away from the cell Good for looking at agents that modulate the channel by working at its intracellular face

Question 27

Applications of whole-cell recordings

Answer 27

Records currents through active channels in the whole cell Good for looking at cell currents in response to drugs added from outside or regulations of channels by the cell Can change solutions fairly rapidly

Question 28

Applications of outside-out cell recordings

Answer 28

Records currents through a singe active channel away from the cell Good for looking at agents that modulate the channel by working at its extracellular face Can change solutions rapidly

Question 29

Mechanism of patch-clamp

Answer 29

- Patch clamp seals can be made on cells in thin slices of brain tissue or even whole brains - Recording is made by applying positive pressure to the electrode - Jet of solution is released from the tip of the electrode to ‘blast’ debris out of the way as the electrode is pushed into the tissue - Technique is typically done ‘blind’ with the neurone hunter relying on changes in electrical resistance to indicate when a cell is under the tip of the electrode - suction is then applied and recordings can be made