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Medsci 317 > Lecture 8; Optogenetics > Flashcards

Lecture 8; Optogenetics Flashcards

1
Q

What is optogenetics?

A

A novel approach to studying synaptic transmission

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2
Q

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

A

1) Reporter
2) Sensory
3) Manipulation / contorl

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3
Q

How can optogenetics function as a reporter?

A

Reporter;

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

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4
Q

How can optogenetics be used as a sensory?

A

Sensor;

  • Dynamic fluorescent sensor of cellular property i.e membrane voltage, Ca2+
  • Fluorescent signal is proportional to cellular property
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5
Q

How can optogenetics be used in manipulation / control?

A
  • Photo activation ledas to change in cellular property i.e membrane potential, synaptic vessel release
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6
Q

Describe how a gene is inserted into the genome;

A

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

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7
Q

What are three methods of gene transfer?

A

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

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8
Q

Describe electroporation

A

High voltage pulses breakdown plasma membrane allowing entry of a plasmid

  • ideal for cell cultures
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9
Q

Describe Stably expressing transgenic animals

A
  • Construct is introduced and incorporated into germ cells

- Colony of expressing animals

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10
Q

Describe viral injection

A

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

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11
Q

Give an example of a gene used in optogenetics reporting;

A

GFP

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12
Q

What is GFP origin?

A

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

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13
Q

How is GFP used?

A
  • 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

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14
Q

What are the applications of GFP protein?

A
  • Target cell type specific recording / observations (whole cell patch clamp recording)
  • Easy visualisation in live tissue (unlike immunihistochemistry that is in fixed tissue)
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15
Q

How does GFP / fluorescence microscopy work?

A
  • 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
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16
Q

What allows optogenetics to be a biosensor?

A

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

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17
Q

What are the advantages of using optogenetics as a biosensor?

A
  • Cell specific targeting

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

18
Q

Describe how optogenetics function as a bio sensor;

A
  • FRET based mechanism

or

  • i.e Voltage depolarisation causes changes in fluorescent signal. Membrane potential is proportional to fluorescence
19
Q

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

A
  • Fast (ms)
  • Heterogenous (many cell types)
  • Highly interconnected (Loops, convergence, divergence)
20
Q

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

A

Electrical stimulation;

  • Fast (good)
  • Spatially imprecise
  • Stimulus artefact

Pharmacological stimulation

  • Slow
  • Dirty, Non-specific actions of drugs

Major problems; Specificity and speed of response

21
Q

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

A

Light is the ideal control

= single cell activation

22
Q

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

A

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

23
Q

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

A

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

24
Q

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

A

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

25
Q

What are the other advantages of optogenetics beyond specificity?

A

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

26
Q

Whats an example of an optogenetics protein being used for ‘control’?

A

Channel Rhodopsin (CHR; ChR2)

27
Q

Describe ChR2;

A
  • Light activated protein

- Physiological nature; Movement function (towards light/energy)

28
Q

Describe the ChR2 structure;

A

7 transmembrane protein

  • Forms an ion channel
  • Fast kinetics
  • Mixed cation conduction (Mostly Na) (inward current)

Activated specifically by blue light (470nm)

29
Q

Describe the mechanism of ChR2;

A

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
30
Q

What does ChR2 enable?

A

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

31
Q

How can animal models be generated for optogenetic investigations?

A

1) Transgenic mouse line

2) On demand animal approach

32
Q

Describe the transgenic mouse line approach;

A

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
33
Q

Describe the on demand animal approach;

A
  • 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

34
Q

What can optogenetic channels be used for?

A

Excitation or inhibition

35
Q

Describe the types of opsins

A 
ChR = Depolarisatiomn
HR = Halorhodopsin = Hyperpolarisation
BR/PR = proton pump (pH)
OptoXR = intracellular signalling
36
Q

What are the limitations of optogenetics?

A

Temporal limitation

Limitations in vivo

37
Q

Describe the temporal limitations of optogenetics;

A
  • 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)

38
Q

Describe the limitations of optogenetics in vivo;

A

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

39
Q

What is the solution to the in vivo limitations?

A

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

40
Q

What is an example of a red shifted halorhodopsin varient?

A

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

41
Q

Describe how optogenetics can be used clincally;

A
  • 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

Medsci 317 (32 decks)

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