Modern Methods in Neuroscience

Question 1

What is a sharp electrode recording? (2)

Answer 1

• Fine tipped glass pipette is inserted into the neuron
• Can inject current and measure changes in membrane potential

Question 2

What are the disadvantages of sharp electrode recordings? (3)

Answer 2

• Can’t measure single channels
• Can’t control the membrane potential (inserting the electrode compromises the membrane so ions can leak out/in)
• Can’t change the solutions inside or outside of the cell being measured

Question 3

How does a single channel patch clamp recording work? (3)

Answer 3

• Fill glass pipette with an ionic solution
• Pipette forms a seal with the membrane
• Currents are measured through the channel present in the area where the pipette touches the membrane

Question 4

How does an inside-out patch clamp recording work? (2)

Answer 4

• Pipette forms a seal with the membrane and the section of membrane is detached from the cell
• The cytosolic surface of the membrane can then be exposed to different solutions

Question 5

How does a whole cell patch clamp recording work? (2)

Answer 5

• Pipette forms a seal with the membrane and more suction is applied to break the membrane so that the pipette is in contact with the inside of the cell
• This allows measurement of current changes in the entire cell

Question 6

How does an outside-out patch clamp recording work? (2)

Answer 6

• Pipette forms a seal with the membrane and the section of membrane is detached from the cell
• The external surface of the membrane can then be exposed to different solutions

Question 7

What are the problems with using fluorescent dyes to label cells? (4)

Answer 7

• Can’t label many cells at once
• Limited ability to label a specific cell type
• Limited ability to label cellular compartments
• Limited ability for live labelling

Question 8

What wavelength of light does GFP absorb?

Answer 8

Blue light at 395nm and 475nm

Question 9

What wavelength of light does GFP emit?

Answer 9

Green light at 508nm

Question 10

How does GCaMP imaging work? (3)

Answer 10

• GFP is modified to be dimmer than usual and fused to M13 and calmodulin
• Conformational change in the proteins in the presence of calcium causes GFP to fluoresce brightly
• Allows you to visualise neuronal activity (increased intracellular calcium in synaptic vesicle exocytosis)

Question 11

How does confocal microscopy work? (2)

Answer 11

• A small pinhole is made between the two lenses
• Any light that is not focussed is rejected

Question 12

What is the benefit of confocal microscopy over widefield microscopy?

Answer 12

Increases spatial resolution

Question 13

How does channelrhodopsin work? (3)

Answer 13

• It is a non-selective cation channel
• Activated by blue light at 460nm
• When blue light shines, channel opens and depolarises the neuron

Question 14

How does halorhodopsin work?

Answer 14

• Chloride channel
• Activated by yellow light at 570nm
• When yellow light shines, channel opens and hyperpolarises the neuron