Optogenetics 3 Flashcards
What is a goal in neuroscience
How brain activity links to function and behaviour
WHat is optogentics?
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
What is an advantage of optogenetics?
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)
What are the core features of optogenetics?
- Light-sensitive proteins: microbial opsins
- 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
What are microbial opsins?
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.
What are the three major classes of these optogenetic molecules?
Archaerhodopsins
Halorhodopsins
Channelrhodopsins
What is Archaerhodopsin?
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.
What is Halorhodopsin?
A chloride pump derived from the halobacterium Natronomonas pharaonis. NpHR actively pumps Cl− ions into cells in response to yellow light.
What is Channelrhodopsin?
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.
What can you use promoters for?
To target where a gene is expressed
How can we express ospin in target cells?
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
What is the first step in optogenetics?
Piece together the genetic construct
Find a promoter to drive expression and a gene encoding opside (light sensitive ion channel?
What is the second step in optogenetics?
Insert construct into virus
What is the third step in optogenetics?
Inject virus into the animal brain; opsin is expressed in targetted neurons
What is the fourth step in optogenetics?
Insert ‘optrode’, ‘fibre-optic’ cable plus electrode
What is the fifth step in optogenetics?
Laser light of specific wavelength opens ion channel in neurons
How can we illuminate specific areas or cells?
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).
How do we get compatible readouts?
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
What can optogenetics be used to study?
Neural function in intact circuits and to alter behaviour in awakw behaving animals
What is a photoelectric artifact?
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.
How does optogenetics compare to electrical stimulation?
Control activity in (genetically) defined cell (sub)populations
Physiological neural activity patterns
Better spatiotemporal resolution
Simultaneous electrophysiological recording is possible
How does optogenetics compare to lesion studies?
Reversible/ temporary
Ability to up- and down-regulate activity within a brain region
Possibility to investigate causal relationships
How does optogenetics compare to pharmacology?
Pharmacology does allow you to define specific cells but optogenetics allow:
Better temporal precision
Possibility to investigate causal relationships
What are further developments of opsins?
-Opsin variants
Opsin variants have been developed – increased channel conductance, increased kinetics, altered spectra
What are further developments of opsins?
-Red-shifted opsins
Red-shifted opsins - enable ‘all-optical’ approach with GCaMP
- increased depth penetration of light (longer λ)
What are further developments of opsins?
-Step-function opsins
Step-function opsins essentially “turn on” excited state with blue light and then “turn off” with green light
What are further developments of opsins?
-Opto-XRs
Opto-XRs - light-sensitive G protein-coupled receptors, optical control of intracellular signalling pathways. Potential to apply in non-excitable cells.
What are potential confounds and weakness of optogenetics?
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)]
What is an example use of optogenetics in Systems Neuroscience? Robinson et al. (2020)
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
What does Robinson et al.’s work show in terms of optogenetics?
Using optogenetics allows you to look at causal mechanisms as you can directly look at specific neurons
What is another example use of optogentics?
-non-neuronsal cells: astrocytes
Perea et al. (2014)
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
What did Paz et al. (2013) find?
Activity guided optical stimulation (‘closed loop’)
