Lecture 11; Optogenetics Flashcards Preview

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Flashcards in Lecture 11; Optogenetics Deck (37)
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1

What is optogenetics?

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

2

What are the three broad functions of an optogenetic protein?

1) Reporter
2) Biosensor
3) Control

3

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)

4

What is a common control protein?

Channelrhodopsin (ChR; ChR2 popular variant)

5

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 )

6

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

7

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

8

Why optogenetics over other mechanisms?

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

9

How else could we control cells?

- Electrically
- Pharmacologically

10

Discuss electrical control

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

11

Discuss pharmacological control;

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

Major problems are specificity and speed of response

12

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

13

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

14

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

15

Why paste the gene behind a promoter?

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

16

How would you get the constructs into a cell?

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

17

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?)

18

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

19

What are the types of animal lines

Transgenic Animal line
On demand animal approach

20

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

21

Describe the on-demand approach;

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

22

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

23

What is patterned illumination?

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

24

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

- Benchtop laser + optical fibre
- Wireless implantable fibre optic coupled device

25

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

- 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)

26

What is the temporal limitation of optogenetics?

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

27

Compare some kinetics of common opsins

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)

28

Describe max frequency between CHR2 and CHeTa?

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

Compare this with ChETA

29

What is a limitation of in vivo optogenetic imaging?

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.

30

What is the purpose of red shifted ChR2?

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