New Imaging Techniques 1 Flashcards
What is optogenetics
Using light to trigger specific nerves in the brain.
How does optogenetics work
Channelrhodopsin is protein fluorescent and an ion channel.
These can be engineered and embedded into specific neurons which when light is shined on them, ti causes the opening of sodium and calcium channels to depolarise nerves and fire APs.
How is channelrhodopsin activated
Blue light
How is halorhodopsin activated
Yellow light
What is halorhodopsin
Halorhodopsin is a light-gated chloride pump found in archae: yellow light activates halorhodopsin pump, chloride enters, nerves hyperpolarise, silences action potentials
Where is channelrhodopsin found
Algae - gene taken from algae and put into neurons in the brain
What benefits do optogenetics have
Converts far more precisely than electrical stimulation
Allows study of circuits in psychiatric disease and stem cell transplants
Both channelrhodopsin and halorhodopsin can work together to activate and silence cells respectively.
Can be controlled over very small time scales
Works faster than drugs
Give some applications of optogenetics
Can be integrated into chimeric receptors to follow specific signalling pathways
Can be used to switch on transgenic genes
Can activate kinases which in turn cause the activation of protein-protein interactions
How are opsin-receptor chimaeras formed
Replace bovine rhodopsin loops with specific adrenergic or serotonin receptors.
When blue light shines - activates the specific signalling pathways linked to these receptors (cAMP pathways)
What role does pharmacology play in optogenetics
Enhances protein-protein interaction specifity.
Give an example of how pharmacology enhances optogenetics
Phototropin 1 is a blue light receptor in plants and stimulated by blue light at the BL-excited light-oxygen voltage sensing domain (LOV2) which activates kinase domains to stimulate proteins
How are transgenes activated in optogenetics
light-switchable transgene systems selectively trigger changes in gene function in specific cells.
LightOn, uses the light dimerization property of Vivid, a light oxygen voltage domain containing protein in circadian clock systems, to form a synthetic light-switchable gene-promoter system.
binds promoters after blue light exposure
rapidly initiate transcription of target transgenes in both mammalian cells and mice
Giving precise spatiotemporal control of genes in a cell type–specific fashion - with light
How can optogenetic be used to treat pain
Through gene therapy, install light-sensitive opsins in nociceptor nerve endings of mice.
blue light through cage floor, the mice react to pain (channelrodopsin)
yellow light blocks nerve impulses block sensation pain (halorodopsin - inhibits)
Also blocks pain perception in mouse model human chronic pain
How is channelrhodopsin used to treat blinded mice
Channelrhodopsin to tranfect retinal ganglion cells in humans blinded by retinitis pigmentosa
Seeking to activate light responsive ganglion cells
Stimulates nerves and some vision
What is the connectome project
similar to mapping the human genome - mapping the entire connections of the human brain is a major goal in neuroscience. This is done using brainbow.
What is brainbow
Brainbow is a process by which individual neurons in the brain can be distinguished from neighbouring neurons using fluorescent proteins. This is managed through loxP and Cre recombinase system.
How does loxP/Cre system form different colours
Essentially the different positions and combinations of loxP sites result in different patterns of transgenes and DNA changes formed by the Cre recombinase. Also, different types of loxP and their orientation can work with different transgene promoters to produce different colours. This results in different colours of expression of the neurons - giving the rainbow.
What colours are used in the brainbow
Red, green, blue - these combine to create different colours of the visual spectrum - this is done through fluorescent transgenes.
inserted multiple copies of the transgenes into the genome. Independent expression from each copy allows a range of colours to result from the combinatorial expression of the XFPs (around 100 colours)
Give an example of when the brainbow has been applied
During development, retinal ganglion cells project to the thalamus and then to the cortex. It was believed that the number of RGCs per thalamic cell decreased through activity-dependent withdrawal, resulting in a 1:1 ratio through regression.
The brainbow tagged all these neurons and found out that these synapses don’t withdraw and die, they actually converge and persist in the thalamus.
Re-evaluated visual circuits
What is one restriction of optogenetics in humans
EXTRA READING - Hickey 2021
Opsins are highly specific and advanced to work efficiently with their native signalling pathways.
These signalling pathways must be changed to function in humans, e.g. a G-protein coupled receptor. This decreases the efficacy of the chimeric opsins massively rendering them inactive/reduced significantly.
Therefore further studies need to be done to try and improve the coupling efficency between the Gprotiens and the opsins
Why is red light preferred for optogenetics
EXTRA READING - boyden 2010
Red light is better tolerated by tissues than light of shorter wavelengths because it scatters less and penetrates more deeply.
What are multicoloured silencers and why are they useful
EXTRA READING - boyden 2010
more powerful and allowed for independent control of multiple kinds of neurons. The multicolor silencers are blue and red—which means that two different neurons can be differentially inhibited by blue light if they bear the two molecules. Boyden’s group described 100 percent neural silencing in mice that were awake.
How are super silencers formed
EXTRA READING - boyden 2010
Arch and Mac genes which code for light-activated proteins and found in bacteria and fungi
How is optogenetics being used to treat people that have went blind with retinitis pigmentosa
They lose peripheral vision and night vision then go blind –>
To overcome the photoreceptor loss, they inect viruses laden with algae DNA into the center of the eye. Their target is the topmost layer of cells in the retina, called ganglions. Once they start making the light-sensitive protein, the ganglion cells should fire in response to light.
