Optogenetics

Question 1

What are channelrhodopsins?

Answer 1

Opsin proteins that act as light-gated ion channels - first discovered in unicellular green algae which displays phototaxis

Question 2

Opsins differ in which 2 ways?

Answer 2

1. Wavelength of light which activates them
2. Kinetics of channel opening/closing

Question 3

Name the 3 main classes of opsins and describe their mechanism of excitation/inhibition.

Answer 3

1. Channelrhodopsins (e.g. ChR2) - cation channel, excitatory
2. Arch - proton pump, inhibitory
3. Halorhodopsins (e.g. NpHR) - chloride pump, inhibitory

Question 4

Name the 2 methods of delivering opsin DNA to a cell.

Answer 4

1. Viral delivery
2. Cre/lox technology

Question 5

Explain how viral delivery is used to insert opsin DNA into a specific cell.

Answer 5

• Opsin DNA is inserted into viral DNA along with a promoter
• The promoter determines which cell types the virus can infect:
  
  • CMV/CAG - all neurons and glia
  • GFAP - glia only
  • hSYN - neurons only
  • CamKII - excitatory neurons only

Question 6

Explain how Cre/lox technology can be used to deliver opsin DNA to specific cells. What are the advantages and disadvantages of using this method?

Answer 6

• Cre recombinase is an enzyme that recognises segments of DNA/mRNA called loxP sites
• Breed two mice: one with ChR flanked by loxP in all cells and one with Cre expressed in specific target cells only
• In progeny, Cre recognises loxP sites facing the same way and deletes stop cassette, enabling translation of ChR protein in target cells only
• Advantage - this allows very specific expression of opsins in target cells
• Disadvantage - process is time-consuming due to breeding

Question 7

Describe the 3 different genetic modifications that can be achieved by Cre recombinase when loxP sites are flanking a gene or DNA segment of interest.

Answer 7

1. Inversion - loxP sites facing eachother, Cre inverts the gene
2. Deletion - loxP sites facing the same way, Cre deletes the gene
3. Translocation - loxP sites on different DNA segments - Cre swaps them around

Question 8

How does optogenetics make it possible to modulate neuronal activity?

Answer 8

• Can excite and inhibit specific cells and regions of the brain depending upon expression of opsins - e.g. cell with ChR2 and NpHR will be excited by blue light and inhibited by yellow light.
• We can mimic real physiology by expressing multiple opsins in the same cell and controlling the wavelength of light applied.
• Potential to inhibit pain, turn on/off emotional responses such as fear.

Question 9

Describe a potential drawback of using the Arch family of opsins as an inhibitory modulator of cells?

Answer 9

• Arch family consists of proton pumps
• Pumping H⁺ out of cells disturbs pH, so may have detrimental effects on normal physiology