Lecture Six: In vivo continued

Question 1

What are the types of cell recordings?

Answer 1

1) Intracellular
2) Extracellular
3) Patch clamp
- Whole cell
- Single channel (cell attached, outside out, inside out)
- Perforated Patch

Question 2

What does the patch actually do?

Answer 2

Creates a circuit that allows the measurement of cell electric characteristics

Question 3

What are the two types of patch clamp measurements?

Answer 3

1) Current clamp (holds current constant and measures voltage)
2) voltage clamp (keeps voltage constant and measures current input to do this)

i.e the recording will be inverted because it measures the input to counteract the actual function of the cell i.e if the cell starts to depolarise, the recording will show repolarisation

Question 4

How is this current constant maintained?

Answer 4

We inject a current that has a composition similar to that of ECF

Question 5

How is a cell patched?

Answer 5

1. Find healthy cell
2. Fill pipette with internal solution and apply positive pressure->To avoid tip blockage!
3. In VC run the test pulse (to get square wave) 4.Pipette offset auto
   - I. To zero the offset
   - II.Check electrode resistance ~ 6-8 MΩ
4. Get pipette near cell
5. Push in until see membrane dimple then release pressure
6. Suck gently to form GigaΩseal!!
7. CpFast Auto
   - I.To eliminate the transients due to the pipette capacitance (Cp)
8. Apply holding voltage (-65mV)
9. Break in by applying brief suction
   - I.See cell capacitance transients

Question 6

Whats the membrane test?

Answer 6

A method of isolating an excitable cell,

Resistance will increase greatly as the pipette tip approaches the membrane therefore the current decreases significantly to achieve the voltage step.

V=IR

Question 7

What does the recording look like when a gigohm is achieved?

Answer 7

Flatline as very little current can exist due to the extreme resistance

Small transients are due to the capacitance of the pipette and these can be dialled out

Question 8

What does the squere wave look like once breakthrough is achieved?

Answer 8

That characteristic upwards spike, decay, stabilised and inflexion of this to baseline.

Question 9

What are some features of a cell you can explore once it has become part of the circuit?

Answer 9

Ra (Access resistance)
Rm (Membrane resistance)
Cm (Membrane capacitance)

Question 10

What is access resistance?

Answer 10

The resistance through the electrode tip into the cell. Often increases during recording as membrane seals back over electrode tip

Question 11

What is membrane resistance?

Answer 11

The resistance through the cell membrane

Depends on cell size
Small cells –high Rm
Large cells –low Rm

Can be used to estimate age of newborn cells

Question 12

What is membrane capacitance dependent on?

Answer 12

The neuronal membrane is a highly efficient capacitor, i.eit stores charge!!!Depends on cell size

Question 13

What are some compensations and corrections for patch clamping?

Answer 13

• Pipette offset
• Pipette capacitance
• Series Resistance
• Slowed responses due to whole cell
• Liquid Junction Potential

Question 14

What are paired recordings?

Answer 14

Recording from two neurons to see if they are synaptically connected based on generating an AP in one and recording depolarisation in the other.