- 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

Lecture 8; Optogenetics Flashcards by Calum Macindoe

What is optogenetics?

A novel approach to studying synaptic transmission

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

1) Reporter
2) Sensory
3) Manipulation / contorl

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How can optogenetics function as a reporter?

Reporter;

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

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How can optogenetics be used as a sensory?

Sensor;

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

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How can optogenetics be used in manipulation / control?

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

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Describe how a gene is inserted into the genome;

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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What are three methods of gene transfer?

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

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Describe electroporation

High voltage pulses breakdown plasma membrane allowing entry of a plasmid

ideal for cell cultures

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Describe Stably expressing transgenic animals

Construct is introduced and incorporated into germ cells

- Colony of expressing animals

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Describe viral injection

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

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Give an example of a gene used in optogenetics reporting;

GFP

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What is GFP origin?

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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How is GFP used?

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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What are the applications of GFP protein?

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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How does GFP / fluorescence microscopy work?

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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What allows optogenetics to be a biosensor?

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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What are the advantages of using optogenetics as a biosensor?

Cell specific targeting

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

Describe how optogenetics function as a bio sensor;

FRET based mechanism

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

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

Fast (ms)
Heterogenous (many cell types)
Highly interconnected (Loops, convergence, divergence)

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

Electrical stimulation;

Fast (good)
Spatially imprecise
Stimulus artefact

Pharmacological stimulation

Slow
Dirty, Non-specific actions of drugs

Major problems; Specificity and speed of response

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

Light is the ideal control

= single cell activation

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

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

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

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

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

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

What are the other advantages of optogenetics beyond specificity?

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

Whats an example of an optogenetics protein being used for 'control'?

Channel Rhodopsin (CHR; ChR2)

Describe ChR2;

- Light activated protein | - Physiological nature; Movement function (towards light/energy)

Describe the ChR2 structure;

7 transmembrane protein - Forms an ion channel - Fast kinetics - Mixed cation conduction (Mostly Na) (inward current) Activated specifically by blue light (470nm)

Describe the mechanism of ChR2;

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

What does ChR2 enable?

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

How can animal models be generated for optogenetic investigations?

1) Transgenic mouse line | 2) On demand animal approach

Describe the transgenic mouse line approach;

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

Describe the on demand animal approach;

- 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

What can optogenetic channels be used for?

Excitation or inhibition

Describe the types of opsins

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

What are the limitations of optogenetics?

Temporal limitation | Limitations in vivo

Describe the temporal limitations of optogenetics;

- 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)

Describe the limitations of optogenetics in vivo;

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

What is the solution to the in vivo limitations?

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

What is an example of a red shifted halorhodopsin varient?

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

Describe how optogenetics can be used clincally;

- 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