New Imaging Techniques 1 Flashcards
Optogenetics
uses light to control cell and animal behaviour
who coined name for optogenetics?
Karl Diesseroth in 2006
phototaxis
algae swim to or away from light
2 properties of algal photoxis protein channelrhodopsin
fluorescent and an ion channel
channelrhodopsin in response to blue light
channel opens, sodium and calcium in, nerve depolarises and fire AP
what is halorhodopsin?
light gated chloride pump found in archae
what activates halorhodopsin?
yellow light - chloride in, hyperpolarise, silence AP
Briefly how does optogenetics work?
Light sensitive protein from algae
take gene for this protein
insert DNA into specific neurons in brain
neurons fire by shining bluelight
Optogenetics - spatial and temporal resolutions
very high spatial - single cell/synapse
high temporal - msec
Opto-XRs
opsin receptor chimeras
What allows chimeric GPCR receptors?
common structure-function relationships
Making Opto-XRs
Replace intracellular loops of bovine rhodopsin with specific adrenergic or serotonin receptors
advantage of Opto-XRs
optically activate intracellular pathways recruited by these receptors eg cAMP and IP3 pathways
What can enhance the specificity of optogenetics?
pharmaoclogy
what is phototropin 1?
blue light (BL) receptor in plants which shows BL dependent kinase activation
What do psychiatric diseases disturb which is a limitation to optogenetics?
intracellular pathways requiring protein-protein interactions
BL on phototropin 1
BL excited LOV2 activates kinase domains
induces protein-protein dimerisations, subsecond and subcellular resolution
Application for light mediated intracellular protein-protein interactions
phototropin 1
What can be used as a light switchable transgene for control of gene expression
Lighton uses light dimerisation property of vivid
vivid
light oxygen voltage domain containing protein in circadian clock systems
forms light switchable gene-promoter system
How to use Light on/vivid
binds promoter after BL exposure
rapidly initiate transcription of target transgene
precise spatiotemporal control of genes in a cell type types specific fashion
2 therapeutic uses of optogenetics
pain
vision
pain - optogenetics
light sensitive opsins in nociceptor nerve endings
blue light - react to pain, yellow light blocks
also blocks pain perception in chronic pain
vision - optogenetics
channel rhodopsin to transfect RGC blinded by retinitis pigmentosa
active light responsive ganglion cells
Cajal
nobel prize 1906, silver nitrate staining in histology
nervous system structure
discovered synapses and growth cones
connectome project
map entire connections of human brain
started 2010
brainbow and connectome project
markers to differentiate between same types of cells
brainbow generated array of fluorescent markers which label each neuron
which system does brainbow use?
cre loxP
where is cre from?
bacteriophage P1
how many fluorescent transgenes does brainbow use?
3/4
RGC projection
thalamus to cortex
1st week of life - how many RGC inputs does 1 thalamic cell recieve? by week 3? what is this called?
20 to 1
1 to 1
activity dependent synapse withdrawl
brainbow and RGC synapse withdrawl
clusters of retinal terminal - label each cell
complex retinothalamic synapses
synapses not withdrawn - convergence persists on thalamic relay cells
