Lecture Five: In vivo patch clamping Flashcards Preview

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Flashcards in Lecture Five: In vivo patch clamping Deck (35)
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1

What is current?

Net ion flow

2

What is voltage?

Potential Energy

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

3

What is resistance/conductance?

Ease of which ions flow

4

What is capacitance?

Storage of charge

5

What rule underlies patch clamping?

Ohms law


V=IR

6

What does capacitance do?

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

7

Whats the problem with recording single channels?

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

8

What solved the noise of a single channel recording?

The giga ohm seal reduced back ground noise

9

What are three variations of patch clamping?

- Perforated whole cell patch clamping
- Whole cell
- Isolated patch

10

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

- Inside out and outside out configurations

11

What are the steps in patch clamping?

- 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

12

What can patch clamping measure?

- 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

13

What do we typically patch clamp?

Neurons


Also cardiac and muscle is less common

14

What is the difficulties of neurons?

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

15

What are some considerations for measuring neurons?

- 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.

16

What are electrophysiological techniques to measure synaptic potential?

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

17

What is a field potential?

–Population potentials (multiple synapses, multiple cells)
–Negative deflection reflects loss of +ve ions. –Easy and quick

18

What is a single whole cell patch clamp?

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

19

What is a dual whole cell patch clamp?

–two cells, few synapses (1 –10 ish)
–Harder still

20

What did patch clamping discover at the synapse?

•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

21

What can you generate to characterise ion channel properties?

I-V curves

22

Whats a IV curve?

- 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

23

What can IV curves infer for pharmaceutical companies?

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

24

Describe the AMPA channel characteristics?

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

25

Hows the IV relationship in most excitable cells?

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


26

What happens when IV relationships are non linear?

Currents are rectified

27

Describe outward and inward rectification;

–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

28

Whats the implication of rectification?

Rectification plays an important role in controlling the excitability of neurons

29

What can determine linear vs non linear IV relationship?

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

30

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

AMPA receptors lacking the GluA2 subunit are inwardly rectifying