Lecture Five: In vivo patch clamping

Question 1

What is current?

Answer 1

Net ion flow

Question 2

What is voltage?

Answer 2

Potential Energy

How much energy between two points (voltage = charge separation (RMP))

Question 3

What is resistance/conductance?

Answer 3

Ease of which ions flow

Question 4

What is capacitance?

Answer 4

Storage of charge

Question 5

What rule underlies patch clamping?

Answer 5

Ohms law

V=IR

Question 6

What does capacitance do?

Answer 6

Slows the change of voltage from square wave format to a sigmoid

Question 7

Whats the problem with recording single channels?

Answer 7

The scale of current is in pico amps and therefore is dominated by background noise

Question 8

What solved the noise of a single channel recording?

Answer 8

The giga ohm seal reduced back ground noise

Question 9

What are three variations of patch clamping?

Answer 9

• Perforated whole cell patch clamping
• Whole cell
• Isolated patch

Question 10

What are a couple of techniques you can use for patch clamping?

Answer 10

• Inside out and outside out configurations

Question 11

What are the steps in patch clamping?

Answer 11

• Electrode within patch clamp
• Positive ion flow out (Current step)

1) Identify a good cell
2) position electrode using indentation on cell because of electrode
3) Induce suction to form the giga ohm seal (resistance is in the ohms)
4) Null the electrode transient (so no transient is present and changes can be detected)
5) Perform break in (kiss)
6) Null the whole cell transient

Check this

Question 12

What can patch clamping measure?

Answer 12

• Voltage step (current is injected to maintain a voltage and this injection matches that going through the channels and thus the voltage can be inferred)
• Current step (change in voltage reflects current)

Check this

Question 13

What do we typically patch clamp?

Answer 13

Neurons

Also cardiac and muscle is less common

Question 14

What is the difficulties of neurons?

Answer 14

• Cannot clamp the entire cell because the processes are too long
• Very fragile and easily killed

Question 15

What are some considerations for measuring neurons?

Answer 15

• Slice preparations vs in vitro preparations
• Importance of cold preparations
• Antagonists to modulate other channels
• Neurons are highly excitable vs glial cells therefore it is easy to ensure you have a neuron.

Question 16

What are electrophysiological techniques to measure synaptic potential?

Answer 16

• Field potentials
• Single whole cell patch clamp
• Dual whole cell patch clamp
• In vivo patch clamp

Question 17

What is a field potential?

Answer 17

–Population potentials (multiple synapses, multiple cells)

–Negative deflection reflects loss of +ve ions. –Easy and quick

Question 18

What is a single whole cell patch clamp?

Answer 18

–One cell, multiple synapses (~ thousands)
–Harder
–Inward currents shown as downward deflections

Question 19

What is a dual whole cell patch clamp?

Answer 19

–two cells, few synapses (1 –10 ish)

–Harder still

Question 20

What did patch clamping discover at the synapse?

Answer 20

• Quantal = release of single vesicle
• Whole cell recordings showed that discrete sized events occurred supporting that transmitter release occurs in “units” (“quanta”) that we now know are vesicles

Question 21

What can you generate to characterise ion channel properties?

Answer 21

I-V curves

Question 22

Whats a IV curve?

Answer 22

• Reveals channel properties
• plots current through a channel as a function of membrane potential change
• Can infer function of channel from this i.e Na and K channels in AP

Question 23

What can IV curves infer for pharmaceutical companies?

Answer 23

Gradient of the slope infers conductance, change in this slope is change in conductance

Where the line crosses 0 reveals reversal potential = where current direction reverses

Question 24

Describe the AMPA channel characteristics?

Answer 24

• Conducts current over a linear range of voltages, reverses at 0

Question 25

Hows the IV relationship in most excitable cells?

Answer 25

In most excitable cells, the current-voltage relation is not always linear (eg: conductance varies with membrane voltage)

Question 26

What happens when IV relationships are non linear?

Answer 26

Currents are rectified

Question 27

Describe outward and inward rectification;

Answer 27

–Outward rectification: a membrane allowing outward current to flow more easily than inward current –Inward rectification: a membrane allowing inward current to flow more easily than outward current

Question 28

Whats the implication of rectification?

Answer 28

Rectification plays an important role in controlling the excitability of neurons

Question 29

What can determine linear vs non linear IV relationship?

Answer 29

Subunit makeup of receptors can determine linear vs non-linear conductances

Question 30

Give an example of a AMPA receptor that is not a linear IV relationship;

Answer 30

AMPA receptors lacking the GluA2 subunit are inwardly rectifying

Question 31

Describe the NMDA receptor IV relationship;

Answer 31

* Very non-linear * Voltage-dependence, sensitive to external Mg2+ * Conductance of the channel changes with voltage

Question 32

Describe the ESPC; (NMDA and AMPA)

Answer 32

* AMPA receptor-mediated EPSCs show fast kinetics * Slow time course of NMDA receptor-mediated EPSCs due to the slow kinetics of the NMDA receptor (slow rise and decay times) - When both are active the decay of the EPSP is prolonged because of the NMDA

Question 33

Why are CNS synapses unreliable?

Answer 33

* One presynaptic terminal, one active zone * One action potential may or may not release the contents of one vesicle •p = ~ 0.1 –0.5 at most CNS synapses –Complicated docking mechanism –Readily releasable pool and reserve pool of vesicles

Question 34

contrast field and patch clamp recordings of potentials;

Answer 34

•Population recordings don’t reflect the true low p properties of CNS synapses as they will consistently report a potential as some cell will fire whilst whole cell recordings show that these firings are very variable.

Question 35

What is current rectification?

Answer 35

This is where a channel favours either inward or outward current direction more than the other (Preference) This results in the bent IV curve. There will be two curves because one is favoured more than the other Reversal is when its linear and no preference.