Optogenetics

Question 1

Optogenetic basiscs for intro

Answer 1

• Based off of the concept of transduction of light stimuli into neuronal signalling via retinal cells —> based on opsins (rods and cones)
• It is possible to isolate the DNA of bacterial opsins that unlike those in the eyes, directly connect to channels rather than the signalling being processed through a biochemical cascade
• Controlling a channel is much easier than having to rely on a complex biochemical cascade in order to control levels of polarization
• Putting these opsins into specific neurons, researchers are able to excite or inhibit neurons directly simply by adding light of a specific frequency

Ed Boyden 2005

• One of the pioneers of optogenetics, Ed Boyden explained in his TED talk that although we have drugs and electric stimulation treatments for brain disorders, both are quite imprecise therefore only alleviating symptoms without curing the actual disorder.
• This led to the idea that if there are molecules in nature that are able to convert light into electricity, then if we can install these proteins into neurons, we can then control this neuron with light, while the neighbouring neurons that do not have the protein would not be affected

Question 2

Optogenetic excitation

Answer 2

• Ed Boyden’s group initially found a type of algae which contains channelrhodopsin
• this is necessary because the algae needs to swim towards to light for photosynthesis, thus must be able to detect light
• does this by use of proteins that can convert light into electricity: channelrhodopsins
• respond to blue light —> opens channels which allow positively charged ions to enter cell —> leads to AP generation

Modifying ChR2

• modifications have been made to create channels with specific neuromodulatory properties
  e. g. extending activation time

—> channels can stay open for several minutes or even hours after one blue light pulse

e.g. altering wavelength

—> creating channels that respond to different colours

—> allows for experiments where activity of multiple cell types can be manipulated at the same time but in different ways

Question 3

Optogenetic inhibition

Answer 3

NpHR (Halorhodopsin)

• Cl- pump
• yellow light —> CL- pumped into cell —> hyperpolarizes cell —> inhibits

Arch (Archaerhodopsin)

• hydrogen pump
• yellow light —> hydrogens pumped out of cell —> hyperpolarizes cell —> inhibits

Limitations of inhibitory opsins***

—> need for continuous light stimuli in order to keep neurons in a hyperpolarized state

—> unlike ChR2 where pulses of blue light are administered, yellow light needs to be delivered continuously

—> if an experiment requires neurons to inhibited for long periods of time, optogenetics is not practical and may come with side effects such as heat causing neural damage

—> instead, probably best to use pharmacological agents for such experiments

Question 4

Administration of Opsin into target neuron

Answer 4

• Channelrhodopsin and other opsins are proteins which are encoded for in the DNA of the organisms
• this DNA can be isolated and taken into a gene therapy vector, most commonly a virus
• neuron is exposed to virus —> starts expressing that opsin —> now neuron can be activated by light
• by tweaking the virus, one can deliver the opsin to specific cells only

Question 5

Methods for delivering light

Answer 5

In Vitro

• light source can be positioned directly next to bath or dish
• or coupled with microscope using specialized shutters

In Vivo

• Challenges
  
  • more difficult —> light must penetrate skull
  • must be delivered very close to target neuron because brain tissue scatters light exponentially
  • delivery system must be light weight to allow free movement
• Common methods
  
  • implanting guide cannula with fibre optic cable
  • LED mounted directly over brain with a cranial glass window
    
    • traditionally only for superficial cortical neurons
    • can combine LED with fibre optic implants to deliver light to deep brain structures —> no need for cables

Question 6

Uses for optogenetics - neural circuits

Answer 6

Basic idea according to Ed Boyden

• use optogenetics to activate neurons to see what powers they unleash, what their activation causes
• deactivate neurons to see what they are necessary for

—> extremely advanced and precise form of lesions

Also possible to study pathways

• can stimulate fibre projections to distinct downstream regions
• e.g. can investigate whether a population of neurons causing a specific phenotype is mediated by a projection to a downstream brain region

Question 7

Neuromodulation in humans

Answer 7

• Optogenetics has obvious therapeutic potential in neurodegenerative diseases and psychiatric disorders
• Could also have applications in neuroenhancement
• BUT applications are highly controversial amongst neuroethicists
• BUT other methods are already used e.g. surgical removal of brain regions to treat epilepsy, electrical stimulation used in PD or severe depression

—> optogenetics have not yet been properly applied to human samples

• difficulties in delivering DNA of opsins as they need to be delivered in vivo
• currently viral gene delivery is the only strategy used to deliver DNA to human neurons
• certain viruses are safe in animal models, but researchers have been reluctant to inject them into humans

Question 8

Limitations - selective expression

Answer 8

• some evidence suggests that not all cells of a certain type will express opsin to the same level —> a specific light intensity will have variable effects on specific cells
• light intensity drops exponentially from the light source (light scatters in the brain) —> needs to be very close to not lose intensity

Question 9

Limitations - Kinetics and synchronization

Answer 9

• ChR2’s channels do not mimic in vivo channels of neurons
• but ChR2 has been successfully modified to have more favourable kinetic properties
• synchronous activation of affected cells makes it difficult to understand how population of cells communicate with one another or how their phasic properties of activation may relate to the circuit —> making all of them fire in the same way?

—> possible to combine optogenetics and fMRI to identify connectome (map of brains’s neural connections)

BUT limited due to spatial and temporal resolution of fMRI not being good enough for studying densely packed and rapid-firing neuronal circuits —> resolution simply not good enough