New Imaging Techniques 1

Question 1

What is optogenetics

Answer 1

Using light to trigger specific nerves in the brain.

Question 2

How does optogenetics work

Answer 2

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.

Question 3

How is channelrhodopsin activated

Answer 3

Blue light

Question 4

How is halorhodopsin activated

Answer 4

Yellow light

Question 5

What is halorhodopsin

Answer 5

Halorhodopsin is a light-gated chloride pump found in archae: yellow light activates halorhodopsin pump, chloride enters, nerves hyperpolarise, silences action potentials

Question 6

Where is channelrhodopsin found

Answer 6

Algae - gene taken from algae and put into neurons in the brain

Question 7

What benefits do optogenetics have

Answer 7

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

Question 8

Give some applications of optogenetics

Answer 8

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

Question 9

How are opsin-receptor chimaeras formed

Answer 9

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)

Question 10

What role does pharmacology play in optogenetics

Answer 10

Enhances protein-protein interaction specifity.

Question 11

Give an example of how pharmacology enhances optogenetics

Answer 11

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

Question 12

How are transgenes activated in optogenetics

Answer 12

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

Question 13

How can optogenetic be used to treat pain

Answer 13

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

Question 14

How is channelrhodopsin used to treat blinded mice

Answer 14

Channelrhodopsin to tranfect retinal ganglion cells in humans blinded by retinitis pigmentosa

Seeking to activate light responsive ganglion cells

Stimulates nerves and some vision

Question 15

What is the connectome project

Answer 15

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.

Question 16

What is brainbow

Answer 16

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.

Question 17

How does loxP/Cre system form different colours

Answer 17

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.

Question 18

What colours are used in the brainbow

Answer 18

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)

Question 19

Give an example of when the brainbow has been applied

Answer 19

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

Question 20

What is one restriction of optogenetics in humans

EXTRA READING - Hickey 2021

Answer 20

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

Question 21

Why is red light preferred for optogenetics

EXTRA READING - boyden 2010

Answer 21

Red light is better tolerated by tissues than light of shorter wavelengths because it scatters less and penetrates more deeply.

Question 22

What are multicoloured silencers and why are they useful

EXTRA READING - boyden 2010

Answer 22

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.

Question 23

How are super silencers formed

EXTRA READING - boyden 2010

Answer 23

Arch and Mac genes which code for light-activated proteins and found in bacteria and fungi

Question 24

How is optogenetics being used to treat people that have went blind with retinitis pigmentosa

Answer 24

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.

Question 25

What is a restriction regarding optogenetics use in blind people

EXTRA READING
Reardon 2016

Answer 25

it responds only to the blue component of natural light, resulting in monochromatic vision.

Question 26

Why is it difficult to brainbow in mammals im comparison to fish

EXTRA READING - Lichtman 2008

Answer 26

Mammals have many more neurons and theyre much denser.
Diversity of neuron types
Long neurons

Question 27

Why could the brainbow be useful in psychiatry disease

EXTRA READING - Lichtman 2008

Answer 27

defects in the pattern, number or proportions of connections might underlie behavioural disorders with a developmental component, such as schizophrenia and autism. By forming the connectome with the brainbow these structural differences can be identified.

Question 28

How can brainbow be used in aging

EXTRA READING - Lichtman 2008

Answer 28

Can determine changes in connectional patterns over an animals age.

Question 29

What other cells can brainbow be used on

EXTRA READING - Lichtman 2008

Answer 29

Other neural cells - astrocytes, Schwann cells and Bergman glia

Question 30

Why is brainbow good at looking at neurons over time

WIDER READING - Weissman 2015

Answer 30

It is inheritable DNA recombination. Therefore an initial pool of progenitor cells that is labeled in specific colors produces labeled cells that reflect their cellular lineage - every mitosis cell after the initial labelled cell will have the same colour.

Question 31

How is colour discrimination being improved with brainbow

WIDER READING - Weissman 2015

Answer 31

By allowing a cell to have two copies of cytoplasmic brainbow and 2 copies of nuclear brainbow, this allows 7 of each. 7 times 7 is 49 meaning that it would be easier to identify a cluster of 7 distinct colours from two different cellular compartments

Question 32

What did brainbow help to find out regarding NMJ nerve injury

WIDER READING - Kang and Lichtman 2013

Answer 32

Nerve cells regenerate faster and more effectively in the old if the debris from the old neurons is cleared in front of them - an area of pharmacological treatment possibly

Question 33

What is diffraction and why does it make dense neural tissue hard to differentiate in brainbow

EXTRA READING - Lichtman 2008

Answer 33

Diffraction - the process by which a beam of light is spread out as a result of passing through a narrow slit/gap.

Because of diffraction, neuronal processes that come closer to each other than approximately 0.25 μm are not resolvable in the light microscope. It is therefore difficult to trace slender axons and dendrites without there being any ambiguities.

Question 34

How did a study use optogenetics to specifically modulate neurons to determine a possible underlying cause for austism and schozophrenia

EXTRA READING - Sohal 2009

Answer 34

Parvalbumins (calcium binding protein) have been believed to cause gamma oscilltations in the brain which enhance information processing but this cannot be selectively controlled. These parvalbumin proteins are believed to cause altered gamma frequency synchronisation in schizophrenia and autism

Optogenetics was able to specifically excite and inhibit parvalalbumin which caused a respective change in gamma oscilltations. Backing up that hypotehsis and opening a door to a new method of assessing brain function and processessing.