Optogenetics

Question 1

What is optogenetics?

Answer 1

The use of genetics to engineer neurons to be responsive to light

Question 2

Where does optogenetics originate from?

Answer 2

Unicellular algae chlamydomonas

Question 3

What channel found in chlamydomonas is used in optogenetics?

Answer 3

Channel rhodhopsin ChR2 - Mixed cation channel with a net influx of cations

Question 4

What is the order of affinity for cations of ChR2?

Answer 4

H+ > N+ > K+ >Ca2+

Question 5

How can ChR2 be expressed in a cell with no other v-gated channels?

Answer 5

Inject RNA into frog oocyte, ion channels are then expressed by transcriptional machinery of cell

Question 6

What effect is seen when ChR2 is clamped at -100mV and +40 mV and exposed to blue light?

Answer 6

-100mV - large initial decrease in current which then increases and plateau
+40 mV - small increase in current before decreasing to a plateau

Question 7

How can the permeability of the ChR2 channel to different ions be measured? What a result of this?

Answer 7

• Measure the current that flows at different voltages in the presence of different cations in the external solution
• channel is not very selective, lithium can pass through just as well as Na

Question 8

How does ChR2 work?

Answer 8

All-trans-retinol in the pore absorbs a proton and changes to 13-cis-retinol which causes a conformational change opening the pore

Question 9

How can channel rhodophsin be inserted into the model organism?

Answer 9

Gal4 (what) - UAS (where)

Question 10

How does the Gal4-UAS system work?

Answer 10

• One fly has Gal4 construct
• Another fly has UAS coupled with transgene
• UAS drives expression of transgene of Gal4 location

Question 11

How do neurons with Gal4-UAS inserted ChR2 respond to light?

Answer 11

Causes continual firing however these decrease over time with sustained light but don’t decrease with pulses

Question 12

What is the disadvantage of optogenetic behaviour experiments?

Answer 12

Often cannot actually know what the neuron is doing

Question 13

What other variations of channel rhodopsin have been obtained through mining diversity?

Answer 13

• Respond to different wavelengths of light

- Can hyperpolarise (outflux of cations or influx of anions)

Question 14

What are the advantages of using red light in optogenetics?

Answer 14

• Passes through tissue more easily

- Insects cannot see lower wavelengths as well