Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Systems Neuroscience > Optogenetics > Flashcards

Optogenetics Flashcards

1
Q

What is optogenetics?

A

The combination of genetic and optical methods to achieve either gain or loss of function of well-defined events in specific cells of living tissue

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What are the advantages of optogenetics?

A

Allows high spatial and temporal control of neural activity

Allows cell specificity

Can be used in many model systems (e.g., fly, zebrafish, rodent)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What are the core features of optogenetics?

A
  1. Light-sensitive proteins = microbial opsins
  2. Ability to target strong and specific expression of opsin genes
  3. Ability to guide light to specific areas/cells with precise timing
  4. Compatible readout approaches
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are microbial opsins?

A

Microbial proteins, known as opsins, are light-activated proteins (channels or pumps) that permit transmembrane movement of ions.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What opsins are used in optogenetics?

A

3 different types:
1. Archaerhodopsins and bacteriorhodopsins
2. Halorhodopsins
3. Channelrhodopsins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is channelrhodopsin?

A

Channelrhodopsins are nonspecific cation channels that depolarize upon blue light illumination

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What are halorhodopsins?

A

Halorhodopsins are light-gated inward chloride pumps which causes hyperpolarization (inhibition) of the cell when triggered with yellow light, thus inhibiting function of the neuron

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What are archaerhodopsins?

A

Also commonly used to inhibit neurons in optogenetic experiments

Arch is a light-activated outward proton pump that hyperpolarizes (inhibits) the cell when triggered by green-yellow light

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What are bacteriorhodopsins?

A

Naturally occurring bacteriorhodopsins, the first-discovered members of this family, which pump protons out of the cell

Bacteriorhodopsin is a protein used by Archaea

It acts as a proton pump; that is, it captures light energy and uses it to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What are the properties of each type of opsin?

A

Archaerhodopsins and bacteriorhodopsins = INHIBITORY
Halorhodopsins = INHIBITORY
Channelrhodopsins = EXCITATORY

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How is selectivity achieved in optogenetics?

A

Express opsin in target cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What methods are used to deliver genes coding for opsin to target cells?

A
  1. Viral delivery: delivery in area injected, promotors target expression to specific neurons
  2. Direct injection into a cre-recombinase transgenic animal
  3. No Injection – breeding of transgenic mouse strains
  4. Specific projections can be targeted with retrograde/anterograde viruses
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What is the typical virus used in viral delivery?

A

Adeno-Associated Virus (AAV)

Very small virus know to infect humans and animals not known to cause any disease

It is possible to ‘load’ up this virus with the genetic information to allow the expression of the opsin once inside the cell of choice

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Explain viral delivery and what typical promotors are used

A

Direct injection of the AAV into the brain : The virus targets only one type of cell by expressing promotors that are only expressed in the cell population of choice

CaMKIIα; biased towards excitatory cells in cortical regions

VGAT will target all GABA inhibitory neurons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Explain direct injection into cre-recombinase transgenic mice

A

You have a transgenic mouse that only expresses an enzyme called cre-recombinase in a specific set of neurons – for example Somatostatin Interneurons.

The AAV will go into all neurons in the injected area but the expression of the opsin will only happen in cells where cre-recombinase is present – extremely well targeted expression in the cell of choice

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Explain breeding of transgenic mice

A

Sst-IRES-Cre knock-in mice express Cre-recombinase in
somatostatin-expressing neurons

Ai32 mice express an improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase

By crossing these two strains of mice 50% of all offspring will have Channelrhodopsin and EYFP in SOM interneurons – ready for blue light stimulation

17
Q

What are the six steps to optogenetics?

A
  1. Piece together genetic construct (promoter to drive expression, gene encoding opsin (light sensitive ion channel))
  2. Insert construct into virus
  3. Inject virus into animal brain, opsin is expressed in targeted neurons
  4. Insert ‘optrode’ fibre optic cable plus electrode
  5. Laser light of specific wavelength opens ion channel in neurons
  6. Record electrophysiological and behavioural results
18
Q

How is cell specificity achieved in optogenetics?

A

LED or laser connected fibre-optic cable - commonly placed in the brain

Local stimulation - position of fibre helps with specificity e.g., can illuminate cell bodies (target all opsin-expressing neurons) or illuminate known downstream region (target projections to that area)

19
Q

What are the compatible readout methods?

A

Need to measure the effect of manipulating neural activity:

  1. Electrophysiology (gold standard - single-cell synaptic input/spiking output)
    ‘Optrodes’ (fibre-optic-electrode hybrid) allow simultaneous electrical readout with optical stimulation
  2. Imaging: Fluorescent indicators (e.g. GECIs) - ‘All-optical’ approach
  3. Behavioural testing
20
Q

What can optogenetics be used to study?

A

Optogenetics can be used to study neural function in intact circuits and to alter behaviour in awake, behaving animals

21
Q

Compared to electrical stimulation, what do optogenetic studies allow for?

A

Control activity in (genetically) defined cell (sub)populations

Physiological neural activity patterns

Better spatiotemporal resolution

Simultaneous electrophysiological recording is possible

22
Q

Compared to lesion studies, what do optogenetic studies allow for?

A

Reversible/ temporary

Ability to up- and down-regulate activity within a brain region

Possibility to investigate causal relationships

23
Q

Compared to pharmacology, what do optogenetic studies allow for?

A

Better temporal precision

Possibility to investigate causal relationships

24
Q

How have opsins been developed further?

A

Opsin variants have been developed - increased channel conductance, increased kinetics, altered spectra

Red-shifted opsins

Step-function opsins

Opto-XRs

25
Q

What do red-shifted opsins allow for?

A

Enable ‘all-optical’ approach with GCaMP

Increased depth penetration of light (longer λ)

26
Q

What do step-function opsins allow for?

A

Essentially ‘turn on’ excited state with blue light and then “turn off” with green light

27
Q

What do Opto-XRs allow for?

A

Light-sensitive G protein-coupled receptors, optical control of intracellular signalling pathways

Potential to apply in non-excitable cells

28
Q

What are the potential confounds of optogenetics?

A

Off-target recombination can occur in Cre lines (e.g. SST-IRES-Cre line): Hu et al. (2013)

Heat from blue light laser can evoke responses in naïve mice [example: fMRI BOLD, Christie et al. (2013)]

29
Q

What did Christie et al. (2013) find regarding heat as a confound of optogenetics?

A

We show that blue light delivery to the naïve rat brain causes profound fMRI responses, despite the absence of optogenetic activation

We demonstrate that these fMRI responses are dependent upon laser power and show that the laser causes significant heating

We identify how heating impacts upon the MR signal causing NMR frequency shifts, and T1 and T2* changes

30
Q

How did Robinson et al. (2020) utilise optogenetics?

A

Investigated the causal role of place cells in spatial navigation

Using an ‘all-optical’ combination of simultaneous two-photon calcium imaging and two-photon optogenetics, we identified and selectively activated place cells that encoded behaviorally relevant locations in a virtual reality environment

31
Q

What were the findings of Robinson et al. (2020)?

A

Targeted stimulation of a small number of place cells was sufficient to bias the behavior of animals during a spatial memory task, providing causal evidence that hippocampal place cells actively support spatial navigation and memory

32
Q

What did Robinson et al. (2020) find about place cell remapping?

A

To assess stimulation-related changes in local network activity, we compared the spatial tuning of place cells in the pre- and post-stimulation epochs. At the single-cell level, neurons remapped, changing their spatial tuning in a variety of ways

This remapping was reflected in a decrease in the single-cell pre-post place map correlation for stimulation sessions

Taken together, these results indicate that targeted optogenetic stimulation of specific place cells triggered remapping of the hippocampal representation of space

Systems Neuroscience (16 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions