Lecture 12 – Optogenetics and Dyes

Question 1

Non-invasive methods to record/ control neuronal activity:

Answer 1

1. Activity reporters (RECORD)
   - What are they?
2. Optogentics (CONTROL)
   - Using light to alter neuron activity

Question 2

Ions move when ion channels open:

Answer 2

E.G. – Ca2+ ions moving through voltage-gated calcium channels
- We can measure the change in Ca2+ using Ca2+ sensitive dyes

Question 3

Calcium sensitive dyes are chelators:

Answer 3

Many different types

• Type used depends on the cell type – EXPERIENCE
  1. Low affinity calcium indicators:
• Excited by UV light
• Examples are Fura-FF, BTC, Fura-2, Fura-5 and Indo-1
  2. Intermediate-affinity calcium:
• Excited by UV light
• Fura-4F
• Fura-5F
• Fura-6F
  3. High affinity and selectivity (BAPTA)
• Excited by visible light under scanning laser confocal microscopy

Calcium green, calcium orange and Oregon green

Question 4

Fluorescence:

Answer 4

Molecular absorption of a photon triggers the emission of another photon with a longer wavelength (which is named after calcium fluoride Fluorite).
They stimulate in the UV range and the emitted light is in the visible range.

Question 5

Example of what can be done with ion-sensitive dyes:

FURA-2 AM LOADED NEURONES (PC12 CELLS)

Answer 5

• black and white shows where nerve cell is
• coloured image of Ca2+ rise induced by adding a neurotransmitter and the hotspots indicate where the neurotransmitter has activated
• the coloured image comes from the Fura-2

Question 6

Other dyes being developed:

Answer 6

1. need to be selective for specific ions
2. can measure Na+, K+ and Cl- but none as good as Ca2+
3. dyes are important because they look at ion fluxes in real time and determine spatial distribution of an ion flux
4. a problem is it still lacks specificity for cell type

Question 7

Genetically encoded Ca2+ indicators [GCaMP]:

Answer 7

latest developments have shown indicators which can be targeted to specific tissues and cells

• can have temporal control
• Ca2+ binding increases fluorescence
• Disadvantages is that’s it’s a relatively weak signal

Question 8

Latest developments:

Answer 8

You can make a transgenic animal that expresses the reporter (e.g. CRISPR)

Question 9

Optogenetics:

Answer 9

Using light to activate ion channels and ion pumps

1. Channel rhodopsin
   - rhodopsin from green algae
   - ion channel
2. halor rhodopsin
   - rhodopsin from halo-bacteria
   - chloride pump

Question 10

Optogenetics to control neuron activity:

Answer 10

• control epilepsy

- parkinson’s disease

Question 11

Optogenetics to control GCPR pathways:

Answer 11

• many signalling pathways in excitable cells activate GCPRs rather than ion channels directly
• can we find light responsive GCPRs to control these pathways with light too?

Question 12

OptoXRs (Optogenetics to control GCPR pathways):

Answer 12

Chimeric (fused) proteins are made of:

• the transmembrane and extracellular domains of the vertebrate rhodopsin
• the intracellular domain of any desired GPCR

Question 13

The future:

Answer 13

• Controlling neurons with magnets or radio waves (magnetogentics)!