Module A - Novel approaches in studying synaptic function with optogenetics

Question 1

What is optogenetics?

Answer 1

Genetically encoded proteins which are either light-activated or fluorescence when excited (by light)
This may occur as proteins fluoresce (photoactivated) when they undergo a conformational change

Question 2

Describe the three broad functions of optogenetics:

Answer 2

1. Reporter (static fluorescent label of cells expressing a reporter gene)
2. Biosensor (dynamic fluorescent sensor of a cellular property, fluorescent signal (output) is proportional to cellular property
3. Control (photolight activation leads to change in cellular property)

Question 3

Describe the steps in light emission:

Answer 3

1. Excitation light (hvEX)
2. S1 energy state is lower than what was put in by the excitation light
3. Emitted light (hvEM) is lower energy, therefore longer wavelength

Question 4

Describe the specificity advantages of optogenetics:

Answer 4

Gene expression under specific cell type promoter (target a single cell type in population of cells)

Location of stimulating light point (light can be focused with laser/microbeam to a small largely definable region, unlike non-specific electrical stimulation)

Different wavelengths (colours) of excitation or emission (multiple optogenic tools can be used concurrently, yet remain discrete)

Light has no off-target effects unlike electrical stimulation

Question 5

Describe the genetic modification advantage of optogenetics:

Answer 5

Customisation of proteins to suit needs (wavelength of activation or excitation or emission, channel permeability, localisation in the cells, kinetics)

Question 6

Describe the light is non-invasive properties of optogenetics:

Answer 6

Although intense light can be damaging to tissue (heat at light focal point)
Although subject to photobleaching

Question 7

Describe the temporal resolution of manipulation of measurement:

Answer 7

Fast (second messengers not greatly required, could be faster)
No artefact from photostimulation unlike electrical stimulation

Question 8

How can promoter-fluorescent tag construct be introduced into cells:

Answer 8

Electroporation
Viral vector
Microinjection

Question 9

Describe the GFP reporter and its applications:

Answer 9

Permits examination of protein-protein interactions

Applications as targeted cell-type specific recording/observations and easy visualisation (live tissue)

Question 10

Describe the components of the optogenetics microscope:

Answer 10

Excitation filter (484nm)
Dichroic mirror >500nm reflected, <500nm transmitted
Emission filter (507nm)

Question 11

Describe the ‘Brainbow’ in optogenetics:

Answer 11

Allows adjacent neurons to be visualised discretely
Utilises random (stochastic) action of Cre-recombinase in Cre-lox system

Question 12

Describe the properties of a biosensor in optogenetics:

Answer 12

Genetically encoded proteins sensitive to voltage, pH, calcium, protein phosphorylation
Same advantages of GFP (cell-specific targeting)
Often FRET-based mechanism

Question 13

Describe the need for precise neuronal control in optogenetics:

Answer 13

Electrical stimulation is not specific

Pharmacological intervention is not specific and slow acting

Question 14

Describe the features of channel rhodopsin (ChR;ChR2 popular variation):

Answer 14

Archetypal optogenetic light activated protein
Isolated from algae (physiological activity - motility towards light/energy and subsequent genetic modifications/improvements)
7 TM protein (forms ion channel with fast cation conductance)

Question 15

Describe the conformation of channel rhodopsin:

Answer 15

Chromophore all-trans-retinal linked to protein
Light causes conformation change to 13-cis-retinal
Subsequent and conformational change to protein (channel opens allowing ions to flow)

Question 16

Describe the result of photo-stimulation of neuron expressing ChR:

Answer 16

Causes inward current (cation flowing into cell), equivalent to membrane depolarisation

Question 17

Describe how generating animals for optogenetics occurs with transgenic mouse lines:

Answer 17

Stably expressing a construct
Cheap to buy ~$700 per breeding pair
Expensive to import and maintain
Inflexibile single promoter and single opsin
Less time intensive (particularly for high use) but still requires careful breeding and regular genotypes (heterozygous offspring)

Question 18

Describe the ondemand animal approach to generating animals for optogenetics:

Answer 18

Intracerebral injection of viral vector (flexible, cheaper than mouse-line)
Variety of cell-type specific promoters
Variety of opsins

Viral vector injection at 3 weeks, 1-6 weeks opsin expression until brain slice, electrophysiology, behavioural studies etc.

Question 19

Describe optogenetics control:

Answer 19

ChR2 enables neuronal activity to be controlled by light selectivity only in those neurons expressing ChR2
Some channels are slow to turn off and may be used for chronic in vivo experiments, but is a limitation in most experiments

Question 20

What is the temporal limitation of optogenetic?

Answer 20

Faster ChR kinetics (shorter time to return to closed state from open state) means higher frequency of reliable action potential stimulation

Question 21

Describe the limitation in vivo of optogenetics:

Answer 21

Blue light is strongly absorbed by blood and scattered by brain tissue resulting in small volume of photoexcited tissue (90% attenuation in 60um)
Red light is absorbed and scattered less, and therefore offers greater volume of photoexcited tissue (requires red-shifted ChR2)
Activate large nuclei in vivo or deep brain structures with superficial light source

Question 22

Describe the limitations in targeting protein to correct cellular location:

Answer 22

Plasma membrane localisation of opsin is ideal (to enable changes in neuronal activity, AP firing rate)
Expression in other membranes (ER/mitochondria) are not ideal
Overcome using genetic toolbox to modify sequence to target opsin to plasma membrane

Question 23

Describe the limitations in sufficient level of expression:

Answer 23

If opsin protein density is too low, insufficient change in membrane potential
Overcome using strong promoters (high endogenous expression) and waiting longer before experimentation starts

Question 24

What is a clinical application of optogenetics?

Answer 24

Parkinson’s Disease