Lecture 3 - methods of research in cellular neuroscience

Question 1

Name the two types of clamp techniques.

Answer 1

voltage clamp - used to measure the current of a cell
current clamp - used to measure the voltage of a cell

Question 2

Describe how voltage clamping was discovered.

Answer 2

The squid giant axon - largest nerve cell in nature.
Researchers threaded electrodes inside the axon.
Could control the insides of the axon - record membrane potential.
As membrane potential changed a current was injected into the electrode to keep the membrane potential stable (clamping the voltage).
Revealed how neurons fire.

Question 3

What were the findings from the squid giant axon experiment?

Answer 3

• whenever an instantaneous current was injected, there was a transient inward current followed. by a delayed outwards current.
• transient inward current reversed at +52mV.
• reversal potential discovered.

Question 4

What is the reversal potential?

Answer 4

when a charged particle can cross a membrane, 2 forces act upon it (electrochemical gradient).
for a given concentration difference you can always find a specific electrical field that provides and equal and opposite force.

Question 5

What can the reversal potential indicate?

Answer 5

• what the concentration is
• if concentration is known, what electrical field is needed to oppose it.

Question 6

Describe current clamping.

Answer 6

used for the analysis of firing patterns.
a cell is taken at rest (no current) and injected a voltage that is kept at a steady state.
this in turn measures what voltage is required to keep it at a steady state.
a closer stimulation of normal neuronal activity.

Question 7

Describe patch clamping.

Answer 7

Sakmann and Neher
Placed a glass electrode down the axon.
Glass will stick onto the side of the cell.
When it sticks a perfect seal with high resistance is created.
Allows you to see sudden step changes as ion channels open/close.
Can see the workings of a single molecule.

Question 8

What was the conclusion made from patch clamping?

Answer 8

Individual recordings appeared jittery.
When averaged out together the recordings resemble that of previous studies.
Conclusion = the whole cell response is equivalent to the average of many individual channels.

Question 9

Phenobarbitone and pentobarbitone significance with patch clamping?

Answer 9

Both enhancers of GABA action.
Able to total how long each channel stayed closed open for through patch clamping.
Conclusion - when barbiturate is given it keeps the channels open but doesn’t change the close time.
Once the channel is open these drugs can keep the channel in the open state for longer.

Question 10

Describe extracellular recordings.

Answer 10

the only recordings used in humans by using a pair of electrodes and measuring the potential between two points.

Question 11

Describe calcium imaging.

Answer 11

Single cell resolution imaging of activity patterns.
Every time an AP is fired a large amount of calcium is taken up from the extracellular space so calcium dyes used to follow the activity with single cell resolution.

Question 12

Describe optogenetics.

Answer 12

using light to control the brain.

Question 13

Chemogenetics - what is a DREADD?

Answer 13

designer receptors exclusively activated by designer drugs.

Question 14

Name 2 examples of DREADDs.

Answer 14

1. Human M3 muscarinic DREADD - coupled to Gq signalling pathway and activates neurons.
2. Human M4 muscarinic DREADD - coupled to Gi signalling pathway and opens K channels/silences neurons (inhibitory).