Lecture 8; Optogenetics

Question 1

What is optogenetics?

Answer 1

A novel approach to studying synaptic transmission

Question 2

What three functionalities (uses) can optogenetics be divided into?

Answer 2

1) Reporter
2) Sensory
3) Manipulation / contorl

Question 3

How can optogenetics function as a reporter?

Answer 3

Reporter;

Static fluorescence label of cells expressing a reporter gene i.e GPCR

Question 4

How can optogenetics be used as a sensory?

Answer 4

Sensor;

• Dynamic fluorescent sensor of cellular property i.e membrane voltage, Ca2+
• Fluorescent signal is proportional to cellular property

Question 5

How can optogenetics be used in manipulation / control?

Answer 5

• Photo activation ledas to change in cellular property i.e membrane potential, synaptic vessel release

Question 6

Describe how a gene is inserted into the genome;

Answer 6

Place gene of interest i.e GFP under a promoter. Promoter could be ubiquitous or cell specific. Therefore end up with GFP only being expressed in cells that youre interested in

Question 7

What are three methods of gene transfer?

Answer 7

1) Electroporation
2) Stably expressing transgenic animals
3) Viral injection

Question 8

Describe electroporation

Answer 8

High voltage pulses breakdown plasma membrane allowing entry of a plasmid

• ideal for cell cultures

Question 9

Describe Stably expressing transgenic animals

Answer 9

• Construct is introduced and incorporated into germ cells

- Colony of expressing animals

Question 10

Describe viral injection

Answer 10

-package viruses with construct of interest, then transduce the cells

Question 11

Give an example of a gene used in optogenetics reporting;

Answer 11

GFP

Question 12

What is GFP origin?

Answer 12

Green Fluorescent Protein
- Isolated from jelly fish
- Genetically modified since then
Allowing for
- Colour variation
- Customised excitation and emission wavelengths
- Improved fluorescence (increased output)

Question 13

How is GFP used?

Answer 13

• GFP tagged onto other proteins of interest. If GFP is detected in a cell then the tagged invisible protein must also be present

Permits examination of protein : protein interactions

Question 14

What are the applications of GFP protein?

Answer 14

• Target cell type specific recording / observations (whole cell patch clamp recording)
• Easy visualisation in live tissue (unlike immunihistochemistry that is in fixed tissue)

Question 15

How does GFP / fluorescence microscopy work?

Answer 15

• Excite GFP with a specific wave length and this will result in the emission (excitation) from the protein of a wavelength lesser than the stimulus

Question 16

What allows optogenetics to be a biosensor?

Answer 16

We can genetically encode proteins to be sensitive to;

• Voltage
• pH
• Ca (i.e cAMP)
• Protein phosphorylation

Thus fluorescence will occur when certain parameters (levels) are reached

Question 17

What are the advantages of using optogenetics as a biosensor?

Answer 17

• Cell specific targeting

- Genetic modification of biosensor to suit needs (speed, wavelength emission, kinetics, intensity)

Question 18

Describe how optogenetics function as a bio sensor;

Answer 18

• FRET based mechanism

or

• i.e Voltage depolarisation causes changes in fluorescent signal. Membrane potential is proportional to fluorescence

Question 19

What are the current problems with studying synaptic transmission and neural microcircuits?

Answer 19

• Fast (ms)
• Heterogenous (many cell types)
• Highly interconnected (Loops, convergence, divergence)

Question 20

What are the current methods of studying synaptic transmission and neural microcircuits?

Answer 20

Electrical stimulation;

• Fast (good)
• Spatially imprecise
• Stimulus artefact

Pharmacological stimulation

• Slow
• Dirty, Non-specific actions of drugs

Major problems; Specificity and speed of response

Question 21

What is the solution to studying synaptic transmission and neural microcircuits?

Answer 21

Light is the ideal control

= single cell activation

Question 22

How is optogenetics used in control / manipulation when studying synaptic transmission and neural microcircuits?

Answer 22

Light activation of selected genetically modified neurons is precise and rapid.

Question 23

What are 1-2 advantages of optogenetics? in terms of specificity

Answer 23

1) Gene expression under specific cell type promoter
- Target a single cell type in a population of cells

2) Location of stimulating light point
- Light can be focused to a small definable region, unlike non specific electrical sitmulation

Question 24

What are 3-5 advantages of optogenetics? in terms of specificty

Answer 24

3) Location of opsin expression (viral vector injection)
4) Different wavelength of excitation or emission
- Multiple optogenetic tools can be used together yet can be distinguished
5) Light has not off target effects

Question 25

What are the other advantages of optogenetics beyond specificity?

Answer 25

1) Specificity 2) Genetic modification (Customisation of proteins to suit needs i.e kinetics, emission,s permeability) 3) Light is non-invasive (but proteins can photo bleach, can heat up at focal point) 4) Temporal resolution of manipulation of measurement (Fast) 5) No artefacts

Question 26

Whats an example of an optogenetics protein being used for 'control'?

Answer 26

Channel Rhodopsin (CHR; ChR2)

Question 27

Describe ChR2;

Answer 27

- Light activated protein | - Physiological nature; Movement function (towards light/energy)

Question 28

Describe the ChR2 structure;

Answer 28

7 transmembrane protein - Forms an ion channel - Fast kinetics - Mixed cation conduction (Mostly Na) (inward current) Activated specifically by blue light (470nm)

Question 29

Describe the mechanism of ChR2;

Answer 29

Chromophore (all trans retinal) linked to protein - Light causes conformational change to 12-cis-retinal - Subsequent conformational cahnge to protein results in the channel opening allowing ion flow

Question 30

What does ChR2 enable?

Answer 30

ChR2 enables neuronal activity to be controlled by light only in those neurons expressing ChR2

Question 31

How can animal models be generated for optogenetic investigations?

Answer 31

1) Transgenic mouse line | 2) On demand animal approach

Question 32

Describe the transgenic mouse line approach;

Answer 32

Transgenic mouse line - Stably expressing a construct - Cheap to buy, but expensive to import / maintain - Inflexible (single promoter, single opsin) - Less time intensive, but requires careful breeding and constant genotyping

Question 33

Describe the on demand animal approach;

Answer 33

- Intracerebral injection of viral vector - Flexible (any target region, promoter) - Cheaper than mouse line - Variety of cell type specific promoters (CAMKII) - Variety of opsins (ChR2, jaws) 1-6 weeks for expression injection 3 weeks post surgery

Question 34

What can optogenetic channels be used for?

Answer 34

Excitation or inhibition

Question 35

Describe the types of opsins

Answer 35

``` ChR = Depolarisatiomn HR = Halorhodopsin = Hyperpolarisation BR/PR = proton pump (pH) OptoXR = intracellular signalling ```

Question 36

What are the limitations of optogenetics?

Answer 36

Temporal limitation | Limitations in vivo

Question 37

Describe the temporal limitations of optogenetics;

Answer 37

- Unable to evoke a spike (AP) for every light stimulus if intensity is too high - Time constraint (T), need 3T for IChR2 to return to baseline - Faster ChR2 kinetics (shorter time to return to closed state) means a higher Hz of AP stimulation (ChR2 =18ms, CHETA = 4ms)

Question 38

Describe the limitations of optogenetics in vivo;

Answer 38

Blue light is strongly absorbed by blood and scattered by brain tissue resulting in a small volume of photo excited tissue

Question 39

What is the solution to the in vivo limitations?

Answer 39

Red light is absorbed and scattered less and therefore offers greater volume of photo excited tissue (requires red shifted ChR2)

Question 40

What is an example of a red shifted halorhodopsin varient?

Answer 40

JAWs is a red shifted halorhodopsin varient. The excitation is similar to conventional halorhodopsin, but the photo current of jaws is 3x greater

Question 41

Describe how optogenetics can be used clincally;

Answer 41

- Parkinsons disease - Loss of dopamanergic neurons in the SNc - Overreactive STN - Imbalance of basal ganglia Trying to use light for brain stimulation not an electrode Viral vectors are the challenge