Lecture Five: In vivo patch clamping Flashcards
What is current?
Net ion flow
What is voltage?
Potential Energy
How much energy between two points (voltage = charge separation (RMP))
What is resistance/conductance?
Ease of which ions flow
What is capacitance?
Storage of charge
What rule underlies patch clamping?
Ohms law
V=IR
What does capacitance do?
Slows the change of voltage from square wave format to a sigmoid
Whats the problem with recording single channels?
The scale of current is in pico amps and therefore is dominated by background noise
What solved the noise of a single channel recording?
The giga ohm seal reduced back ground noise
What are three variations of patch clamping?
- Perforated whole cell patch clamping
- Whole cell
- Isolated patch
What are a couple of techniques you can use for patch clamping?
- Inside out and outside out configurations
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
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
What do we typically patch clamp?
Neurons
Also cardiac and muscle is less common
What is the difficulties of neurons?
- Cannot clamp the entire cell because the processes are too long
- Very fragile and easily killed
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.
What are electrophysiological techniques to measure synaptic potential?
- Field potentials
- Single whole cell patch clamp
- Dual whole cell patch clamp
- In vivo patch clamp
What is a field potential?
–Population potentials (multiple synapses, multiple cells)
–Negative deflection reflects loss of +ve ions. –Easy and quick
What is a single whole cell patch clamp?
–One cell, multiple synapses (~ thousands)
–Harder
–Inward currents shown as downward deflections
What is a dual whole cell patch clamp?
–two cells, few synapses (1 –10 ish)
–Harder still
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
What can you generate to characterise ion channel properties?
I-V curves
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
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
Describe the AMPA channel characteristics?
- Conducts current over a linear range of voltages, reverses at 0
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)
What happens when IV relationships are non linear?
Currents are rectified
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
Whats the implication of rectification?
Rectification plays an important role in controlling the excitability of neurons
What can determine linear vs non linear IV relationship?
Subunit makeup of receptors can determine linear vs non-linear conductances
Give an example of a AMPA receptor that is not a linear IV relationship;
AMPA receptors lacking the GluA2 subunit are inwardly rectifying
Describe the NMDA receptor IV relationship;
- Very non-linear
- Voltage-dependence, sensitive to external Mg2+
- Conductance of the channel changes with voltage
Describe the ESPC; (NMDA and AMPA)
- 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
Why are CNS synapses unreliable?
- 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
contrast field and patch clamp recordings of potentials;
•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.
What is current rectification?
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.
