Lecture 11; Optogenetics

Question 1

What is optogenetics?

Answer 1

Genetically encoded proteins which fluoresce when excited (by light) or are light-activated

Question 2

What are the three broad functions of an optogenetic protein?

Answer 2

1) Reporter
2) Biosensor
3) Control

Question 3

How do optogenetic proteins control a cell (broad definition)?

Answer 3

•Photo (light)-activation leads to change in cellular property (e.g. membrane potential, synaptic vesicle release)

Question 4

What is a common control protein?

Answer 4

Channelrhodopsin (ChR; ChR2 popular variant)

Question 5

Where did the channel rhodopsin come from?

Answer 5

• 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 )

Question 6

Describe the ChR characteristics

Answer 6

• 7TM protein
• Forms Ion channel (atypical usually GCPR)
• Fast Kinetics
• Mixed cation conductance
  
  • Inward flux = depolarisation
• Activated by blue light typically 470nm

Question 7

What is the mechanism of activation for CHR?

Answer 7

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

Question 8

Why optogenetics over other mechanisms?

Answer 8

• Specificity
• Light is non-invasive
• Temporal resolution of manipulation
• No artefact associated with photo-stimulation

Question 9

How else could we control cells?

Answer 9

• Electrically

- Pharmacologically

Question 10

Discuss electrical control

Answer 10

• Fast response (millisecond)
• Stimulus artefact (when recording electrical activity)
• Non-specific

Question 11

Discuss pharmacological control;

Answer 11

• Slow response (>minutes)
• Of f-target (non-specific) effects

Major problems are specificity and speed of response

Question 12

Why is optogenetics so specefic?

Answer 12

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

Question 13

Advantages of optogenetics continued

Answer 13

• 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

Question 14

What are common control opsins?

Answer 14

ChR2 (Na) and HR (Cl-) are commonly used opsins for excitation and inhibition of neuronal activity respectively

As well as;

• Proton pumps
• Intracellular signalling

Question 15

Why paste the gene behind a promoter?

Answer 15

The promotor enables cell-type specific expression of the protein of interest

Question 16

How would you get the constructs into a cell?

Answer 16

1) Electroporation
2) Stably expressed genetic transgenic animals
3) Viral Injection

Question 17

Describe electroporation;

Answer 17

• 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?)

Question 18

Describe transgenic animal line and viral vector;

Answer 18

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

Question 19

What are the types of animal lines

Answer 19

Transgenic Animal line

On demand animal approach

Question 20

Describe transgenic animal line;

Answer 20

• 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

Question 21

Describe the on-demand approach;

Answer 21

• Intracerebral injection of viral vector (stereotaxic)
• Flexible and cheap
• Time intesive
• Variety of promoters and opsins

Question 22

What are the types of illumination for in vitro (cell) preparations;

Answer 22

• Mercury lamp with filter
• Laser (precise and powerful)
• LED (cheap and less power)
• Patterned illumination

Question 23

What is patterned illumination?

Answer 23

• Digital mirror device (>600,000 individual mirrors )
• Enables Functional Mapping technique (covered shortly)
• Connects to microscope

Question 24

What are the types of illumination for in vivo? (animal)?

Answer 24

• Benchtop laser + optical fibre

- Wireless implantable fibre optic coupled device

Question 25

Whats so good about the wireless implantable fibre optic coupled device?

Answer 25

• Wireless control (and optional data transfer)
• Wireless charging of internal battery
• No risk of infection
• Chronic studies
• Full range of behavioural tests (Morris water maze etc)

Other wireless devices are less eloquent (e.g. variable light intensity output, large and restrictive additional hardware)

Question 26

What is the temporal limitation of optogenetics?

Answer 26

Unable to evoked a spike (action potential) for every light stimulus if frequency is too high (refractory like period)

Need 3x τor to return to (nea rly) baseline

Question 27

Compare some kinetics of common opsins

Answer 27

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

ChR2 (H134R): common variety of ChR2 used (18ms)
ChETA: a modified ChR2 with improved (faster) kinetics (4ms)

Question 28

Describe max frequency between CHR2 and CHeTa?

Answer 28

Need 3x τ(3x 18ms ) to return to baseline Max frequency = 1/3τ= ~16 Hz

Compare this with ChETA

Question 29

What is a limitation of in vivo optogenetic imaging?

Answer 29

Red and blue light pass through (transmit) water equally well and with gradual loss over distance

Light Intensity vs Distance

But

IN biological tissue red light offers a greater volume of tissue i.e is transmitter better

Your sensors have to be very close in either instance.

Question 30

What is the purpose of red shifted ChR2?

Answer 30

Red light offers greater volume of photo-excited tissue.

Requires ‘red-shifted’ ChR2
•‘ReachChR’ (617nm excitation)
•Activate large nuclei in vivoor deep brain s tructures with s uperficial light source

Question 31

What is a mapping technique / his area of research?

Answer 31

CRACM

Circuit Mapping ChR2-Assisted Circuit Mapping

Question 32

What is the essence of CRACM?

Answer 32

Mapping synapses from a primary neuron

Question 33

Why do you need optogenetic control for circuit mapping?

Answer 33

Neural networks:

• Highly interconnected (loops, convergence, divergence)
• Fast (millisecond timescale)
• Heterogeneous (many cell types)

Question 34

How does CRACM work?

Answer 34

• reveals functioning connections (synapses) between neurons
• Record electrophysiological response from one neuron
• Optically activates the presynaptic neurons (in a grid like pattern i.e like battleships)

Builds on intensity plot that indicates synapses using algorthims

2D currently

Question 35

Why is the hindrance for using optogentics in the clinic?

Answer 35

How to get expression of opsins in the brain?-

• Gene therapy is possible but viruses have ethical concerns
• Electroporation approach (think cochlear example –no virus required)

Cytotoxic effects?

• Light delivery
• Implantable LED device

Question 36

What are optogentic inspired clinical applications?

Answer 36

TMS

Use optogenetic res earch in the lab to inform/design clinical interventions

Question 37

What are optogentic design experiment ideas?

Answer 37

Target area (viral injection target)
Target cell-type (promotor selection)
Activate or inhibit (opsin selection)
Fast or slow (opsin selection)
Illumination type (in vitro vs  in vivo options )
Limitations to overcome(opsin selection)
Combine with other approaches
Electrophysiology or behavioural output
Immunocytochemistry to validate cell-type expression