Optogenetics and Dyes

Question 1

What is the underlying principle of imaging?

Answer 1

• ions move when ion channels open

- so you can measure e.g. changing calcium as a proxy for neuronal activities

Question 2

What are Calcium sensitive dyes called?

Answer 2

chelators

Question 3

What are the different type of Calcium indicators?

Answer 3

• Low affinity calcium indicators – excited by UV light
• Intermediate-affinity calcium indicators – excited by UV light
• High-affinity and selectivity (BAPTA) – excited by visible light under scanning laser confocal microscopy

Question 4

What is fluorescence?

Answer 4

• Is the phenomenon in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength (named after calcium fluoride ‘fluorite’
• Stimulate in the ultraviolet range, and the emitted light is in the visible range

Question 5

What is Fura-2 an example of?

Answer 5

• an ion sensitive dye

- Negatively charged groups bind to positively charged calcium ions

Question 6

What do Fura dyes do?

Answer 6

• Can measure calcium given the amount of fluorescent given out
• The Fura dye gives the amount of calcium by shifting the peak from 380 to 340 nm and also the amplitude at 340 nm shows how much calcium is present
• You can see calcium entry at particular synapses and sites

Question 7

How do you get over the problem that Fura dyes are hydrophilic and can’t cross the cell membrane?

Answer 7

• chemists put an acetoxymethyl ester onto Fura, makes it hydrophobic
• In the cell there are esterases that break down the ester group
• Just left with Fura-2 in the cell which can’t re-cross the cell membrane

Question 8

Apart from Calcium dyes what other dyes have been developed?

Answer 8

So far can measure Na+, K+, Cl- but none as good as Ca2+ dyes

Question 9

Why are dyes so important?

Answer 9

• Looks at ion fluxes in real-time

- Determine spatial distribution of ion flux

Question 10

What is the problem with dyes?

Answer 10

lacks specificity for cell type

Question 11

What is GCaMP?

Answer 11

• genetically encoded Ca2+ indicator
• Latest developments: can be targeted to individual tissues/ cells/ domains
• Can have temporal control
• Ca2+ binding increases fluorescence
• Disadvantages: relatively weak signal (but camera good enough now that that doesn’t matter)
• GFP has been modulated to contain two calcium binding sites, when calcium is there it binds to CaM domain, this swings towards M13 domain and Ca2+ binding causes increase in fluorescence output
• A ‘transgenic’ animal could expresses the reporter (e.g. CRISPR) – cell specific as it will only be turned on in cells you want

Question 12

What does Optogenetics involve?

Answer 12

manipulating neuronal behaviour using light sensitive ion channels and ion pumps

Question 13

In addition to experimental applications what else does optogenetics have the potential to make an impact in?

Answer 13

Disease therapy

Question 14

What is Channelrhodopsin (ChRH134R)?

Answer 14

• Rhodopsin from green algae (480 nm wavelength) ion channel
• Has a retinal cofactor which works with an ion channel that is permeable to sodium and potassium
• When a blue light photon is absorbed by the retina is causes the opening of an ion channel and sodium goes down it’s sodium gradient to depolarise the cell leading to an action potential

Question 15

What is Halorhodopsin (NpHR)?

Answer 15

• Rhodopsin from halobacterial (590 nm wavelength) chloride pump
• Absorbs a photon of orange light
• Pumps chloride from high concentration to low concentration
• Usually pumped into cell and depolarises it

Question 16

How is light delivered to deep brain regions?

Answer 16

region an optical fibre is usually implanted close to the cells of interest. For surface brain regions LEDs might be used

Question 17

What is a common way that light responsive proteins used in optogenetics are targetted to cells?

Answer 17

via viral infection. An alternative method is to introduce the opsin genes into the animal’s germ line and produce a transgenic animal

Question 18

What diseases can you use optogenetics?

Answer 18

• You can use light to regulate parkinsonian motor behaviours
• You can use it in epilepsy

Question 19

Can you use optogenetics to control GPCR pathways and why would you want to do this?

Answer 19

• Many signalling pathways in excitable cells activate GPCRs rather than ion channels directly – dopamine receptors, serotonin receptors
• Can we find light responsive to GPCRs to control these pathways with light too?
• Chimeric (fused) proteins made of
   The transmembrane and extracellular domains of vertebrate rhodopsin
   The intracellular domain of any desired GPCR

Question 20

What have labs been investigating seeing as light doesn’t go through the skull?

Answer 20

• Labs are playing with controlling neurons with magnets or radio waves (magneto genetics)
• Used mechanosensitive channels which are stretch sensitive
• GFP-ferritin put on protein – put magnetic waves on this which stretches the protein and therefore opens the cell
• Hasn’t been much follow-up with this though