Optogenetics 3

Question 1

What is a goal in neuroscience

Answer 1

How brain activity links to function and behaviour

Question 2

WHat is optogentics?

Answer 2

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

It gives great cell speicificty that was hard to do before optogenetics

Question 3

What is an advantage of optogenetics?

Answer 3

Allows high spatial and temporal control of neural activity

One of the most important recent advances e..g since fMRI it has created a simialr revolution

Can combine many techniques e.g. electrophysiology

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

Question 4

What are the core features of optogenetics?

Answer 4

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

Question 5

What are microbial opsins?

Answer 5

The universal photoreceptor molecules of all visual systems in the animal kingdom. They can change their conformation from a resting state to a signalling state upon light absorption, which activates the G protein, thereby resulting in a signalling cascade that produces physiological responses.

Question 6

What are the three major classes of these optogenetic molecules?

Answer 6

Archaerhodopsins
Halorhodopsins
Channelrhodopsins

Question 7

What is Archaerhodopsin?

Answer 7

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.

Question 8

What is Halorhodopsin?

Answer 8

A chloride pump derived from the halobacterium Natronomonas pharaonis. NpHR actively pumps Cl− ions into cells in response to yellow light.

Question 9

What is Channelrhodopsin?

Answer 9

is a light-activated cation channel capable of inducing depolarization and action potentials in neurons.

Channelrhodopsins are nonspecific cation channels that depolarize upon blue light illumination.

Question 10

What can you use promoters for?

Answer 10

To target where a gene is expressed

Question 11

How can we express ospin in target cells?

Answer 11

Need to deliver genes coding for opsin to target cells. Common methods:

Viral delivery: delivery in area injected, promoters target expression to specific neurons

Cre-Recombinase: transgenic mouse expressing Cre recombinase under cell specific promoter, inject with recombinase-dependent opsin virus.

Transgenic mouse: breed Cre mouse X recombinase-dependent opsin mouse (e.g. Ai32 [ChR2])

Specific projections can be targeted with retrograde/anterograde viruses

Question 12

What is the first step in optogenetics?

Answer 12

Piece together the genetic construct

Find a promoter to drive expression and a gene encoding opside (light sensitive ion channel?

Question 13

What is the second step in optogenetics?

Answer 13

Insert construct into virus

Question 14

What is the third step in optogenetics?

Answer 14

Inject virus into the animal brain; opsin is expressed in targetted neurons

Question 15

What is the fourth step in optogenetics?

Answer 15

Insert ‘optrode’, ‘fibre-optic’ cable plus electrode

Question 16

What is the fifth step in optogenetics?

Answer 16

Laser light of specific wavelength opens ion channel in neurons

Question 17

How can we illuminate specific areas or cells?

Answer 17

LED or laser connected fibre-optic cable - commonly placed in 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).

Question 18

How do we get compatible readouts?

Answer 18

Need to measure the effect of manipulating neural activity:

Electrophysiology (gold standard - single-cell synaptic input/spiking output)

‘Optrodes’ (fibre-optic-electrode hybrid) allow simultaneous electrical readout with optical stimulation

Imaging: Fluorescent indicators (e.g. GECIs) - ‘All-optical’ approach
Behavioural testing

Question 19

What can optogenetics be used to study?

Answer 19

Neural function in intact circuits and to alter behaviour in awakw behaving animals

Question 20

What is a photoelectric artifact?

Answer 20

The photoelectric effect is the process whereby the energy from electromagnetic radiation, such as visible light, gamma radiation or other, hits an atomic electron whereby the energy of the radiation is transferred in its entirety to the electron causing the electron to be ejected from the atom.

Question 21

How does optogenetics compare to electrical stimulation?

Answer 21

Control activity in (genetically) defined cell (sub)populations
Physiological neural activity patterns
Better spatiotemporal resolution
Simultaneous electrophysiological recording is possible

Question 22

How does optogenetics compare to lesion studies?

Answer 22

Reversible/ temporary
Ability to up- and down-regulate activity within a brain region
Possibility to investigate causal relationships

Question 23

How does optogenetics compare to pharmacology?

Answer 23

Pharmacology does allow you to define specific cells but optogenetics allow:
Better temporal precision
Possibility to investigate causal relationships

Question 24

What are further developments of opsins?
-Opsin variants

Answer 24

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

Question 25

What are further developments of opsins?
-Red-shifted opsins

Answer 25

Red-shifted opsins - enable ‘all-optical’ approach with GCaMP
- increased depth penetration of light (longer λ)

Question 26

What are further developments of opsins?
-Step-function opsins

Answer 26

Step-function opsins essentially “turn on” excited state with blue light and then “turn off” with green light

Question 27

What are further developments of opsins?
-Opto-XRs

Answer 27

Opto-XRs - light-sensitive G protein-coupled receptors, optical control of intracellular signalling pathways. Potential to apply in non-excitable cells.

Question 28

What are potential confounds and weakness of optogenetics?

Answer 28

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)]

Question 29

What is an example use of optogenetics in Systems Neuroscience? Robinson et al. (2020)

Answer 29

Head fixed, rodent runs along a VR track to sugary water (lick rate was measured)

Targeted activation of specific place cells produces changes in behaviour and specifically spatially associated behaviour

Halfway they stimulated reward cells to see if it changed behaviour

Question 30

What does Robinson et al.’s work show in terms of optogenetics?

Answer 30

Using optogenetics allows you to look at causal mechanisms as you can directly look at specific neurons

Question 31

What is another example use of optogentics?
-non-neuronsal cells: astrocytes
Perea et al. (2014)

Answer 31

ChR2 stimulation evoked Ca2+ signal in astrocytes
Astrocytes modulated spontaneous activity in PV, SST and excitatory neurons
Astrocytes induced changes in visual responses of PV, SST and excitatory neurons

Question 32

What did Paz et al. (2013) find?

Answer 32

Activity guided optical stimulation (‘closed loop’)