Lecture 11; Optogenetics Flashcards
What is optogenetics?
Genetically encoded proteins which fluoresce when excited (by light) or are light-activated
What are the three broad functions of an optogenetic protein?
1) Reporter
2) Biosensor
3) Control
How do optogenetic proteins control a cell (broad definition)?
•Photo (light)-activation leads to change in cellular property (e.g. membrane potential, synaptic vesicle release)
What is a common control protein?
Channelrhodopsin (ChR; ChR2 popular variant)
Where did the channel rhodopsin come from?
- Isolated from single cell green algae Chlamydomonas reinhardtii(2001)
- Subs equent genetic modifications /improvements
- Physiological activity in nature –movement function (towards light to maintain photosynthesis )
Describe the ChR characteristics
- 7TM protein
- Forms Ion channel (atypical usually GCPR)
- Fast Kinetics
- Mixed cation conductance
- Inward flux = depolarisation
- Activated by blue light typically 470nm
What is the mechanism of activation for CHR?
Chromophore ‘all-transretinal’ linked to protein
•Light causes conformational change to ‘13-cis-retinal’
•Subsequent conformational change to protein –channel opens allowing ions to flow
Why optogenetics over other mechanisms?
- Specificity
- Light is non-invasive
- Temporal resolution of manipulation
- No artefact associated with photo-stimulation
How else could we control cells?
- Electrically
- Pharmacologically
Discuss electrical control
- Fast response (millisecond)
- Stimulus artefact (when recording electrical activity)
- Non-specific
Discuss pharmacological control;
- Slow response (>minutes)
- Of f-target (non-specific) effects
Major problems are specificity and speed of response
Why is optogenetics so specefic?
Specificity:
- Genes expressed under a single promoter (target single cell population)
- Localisation of light source (Light can be focused to very specific area unlike electric stim)
- Location of opsin expression (viral vector)
- Different excitation and emission spectra (multiple optogenetic tools can be used concurrently, yet remain discrete)
- Light has no off target effects unlike pharma
Advantages of optogenetics continued
- Genetic modification (customisation of proteins to suit need)
- Light is non-invasive (Although intense light can be damaging, Heat, photo bleaching)
- temporal resolution of manipulation of measurement (fast, secondary messangers generally not required) (many variations in speed)
- No artefact
What are common control opsins?
ChR2 (Na) and HR (Cl-) are commonly used opsins for excitation and inhibition of neuronal activity respectively
As well as;
- Proton pumps
- Intracellular signalling
Why paste the gene behind a promoter?
The promotor enables cell-type specific expression of the protein of interest
How would you get the constructs into a cell?
1) Electroporation
2) Stably expressed genetic transgenic animals
3) Viral Injection
Describe electroporation;
- High voltage pulse breaks down plasma membrane and allows entry of plasmid
- Ideal for cell cultures
i.e Recent research uses cochlear implant (hearing) to electroporate nearby neurons to express GDNF (possible application for humans and DBS?)
Describe transgenic animal line and viral vector;
Transgenic; Construct is introduced and incorporated in germ cells
Viral vector; Use natures machinery to do the work. Package virus with construct of interest, then transduce cells
What are the types of animal lines
Transgenic Animal line
On demand animal approach
Describe transgenic animal line;
- Stably expressing single construct
- cheap to buy breading pair
- Expensive to import, maintain, feed, genome checks,
- Inflexible as single promoter and opsin
- Less time intensive
- Many species available
Describe the on-demand approach;
- Intracerebral injection of viral vector (stereotaxic)
- Flexible and cheap
- Time intesive
- Variety of promoters and opsins
What are the types of illumination for in vitro (cell) preparations;
- Mercury lamp with filter
- Laser (precise and powerful)
- LED (cheap and less power)
- Patterned illumination
What is patterned illumination?
- Digital mirror device (>600,000 individual mirrors )
- Enables Functional Mapping technique (covered shortly)
- Connects to microscope
What are the types of illumination for in vivo? (animal)?
- Benchtop laser + optical fibre
- Wireless implantable fibre optic coupled device
