Optogenetics

Question 1

What is the basic structure of a neuron?

Answer 1

Axons synapse onto dendrites

• electrical signal is turned into AP
• moves down axon hillock, down axon and synapses onto another neuron, releasing NT to transmit the signal.

Question 2

What is the membrane potential?

Answer 2

The voltage (difference in electrical charge) across the plasma membrane

Question 3

Why is RMP usually -70mV?

Answer 3

We have a high IC K+ concentration and a high EC Na+ concentration.

• K+ exits through channels down its concentration gradient
• 3Na+/2K+ exchanger keeps the cell at -70mV

Question 4

What are the steps in AP initiation?

Answer 4

• resting state (-70mV, VGK/Na channels closed
• stimulus
• Depolarisation (Net positive flow into neuron)
• Threshold reached (-50mV - VGNa channels open)
• VGK channels open, K+ efflux (repolarisation)
• Hyperpolarisation

Question 5

What are light-activated proteins?

Answer 5

• instead of a ligand binding to receptors, light acts on it - APs are transmitted when light comes in (rods and cones do this)

Question 6

How can we get neurons to produce Channelrhodopsin on their surface?

Answer 6

• Insert genes for channelrhodopsin into virus
• these can insert the channelrhodopsin DNA into the cells of the neuron
• neuron can then express channelrhodopsin on its surface

Question 7

What are the three different types of opsins we can use?

Answer 7

• Channelrhodopsins (cation channels - depolarisation/excitation)
• Arch (proton pump - hyperpolarisation/ inhibition)
• Halorhodopsins (Chloride pump - hyperpolarisation/inhibition)

Question 8

What wavelengths activate each of the three opsins?

Answer 8

ChR2 - blue
Arch - green
NpHR - yellow

Question 9

Which promoters determine which cell types? (CMV/CAG, GFAP, hSYN,CamKII)

Answer 9

• CMV/CAG = All neurons and glia
• GFAP = Glia only
• hSYN = Neurons only
• CamKII = Excitatory neurons only

Question 10

What do the different arrangements of LoxP do?

Answer 10

• if LoxPs are facing each other, gene inbetween will be flipped around (may become transcribable/un-transcribable
• if LoxPs are facing in the same direction, then genes inbetween will be excised.
• If LoxPs are on two different sections of DNA, Cre will swap them around

Question 11

What does Cre recombinase do?

Answer 11

Recognises LoxP sites

Question 12

How does the CreLox system work?

Answer 12

• Create one mouse with ChR inserted into genome - flanked with two LoxP sites facing each other
• Stop codon upstream means that ChR wont be expressed
• Create another with Cre recombinase that is only expressed in specific cells
• When these two mate, the first generation mouse will be able to express ChR because the Cre excises the STOP codon upstream

Question 13

How can you modulate neuronal activity using opsins?

Answer 13

• Can use a blue light to open ChR and cause cation influx - depolarisation
• Can use yellow light to activate halorhodopsins and cause chloride influx - hyperpoalrisation

Question 14

2 good and 2 bad things about using optogenetics in humans

Answer 14

Good

• faster than drugs
• more specific than electrical stimulation

Bad

• only gene therapy approved in Europe
• Would we recognise the opsins as non-self and start an immune response?