Module 13 - optogenetics, dues and radioligands Flashcards

1
Q

fluorescence how

A

Absorb high energy light
Then emit light at LONGER wavelength at lower energy

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2
Q

why reporter dyes better than electrophys

A
  • not invasive, no cells destroyed
  • electrophys has limits to what we can patch onto
  • reporter dyes can do multiple cells at a time
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3
Q

what are 3 types of calcium sensitive dyes

A

low affinity = excited by UV light
intermediate affinity = excited by UV light
high affinity = excited by visible light

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4
Q

how does fura-2 bind calcium

A

has 4 COOH- groups
specific to divalent ions ie Ca2+

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5
Q

how does fura-2 change after Ca2+ bound

A

causes distortion
cahnges excitation wavelength

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6
Q

how would you use fura dye to measure conc of Ca2+

A

excite sample at 340nm then 380nm
measure emmision at 510nm for each
- 340 gives conc of Fura2 that has Ca2+ bound to it
- 380 gives Fura2 that isnt bound to Ca2+
Then take the ratio: emm from 340/ emm from 380

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7
Q

what 2 things are affected when Ca2+ added to a sample

A

the emmision peak increases in magnitude, but remains at 510
the absorption shifts from 380 to 340

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8
Q

what changes Fura from polar to non-polar

A

esterify it with Acetoxymethyl Ester (AM ester)
to make Fura-2 AM

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9
Q

problem with Fura 2 AM

A

crosses the memb but can bind Ca2+ cuz all COOHs are esterified
so in the cell, esterases act to remove AM

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10
Q

2 main advantages of these dyes

A

look at ion fluxes in real time
show spatial distribtuion of ion flux

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11
Q

disadvantage of dye

A

lacks specificity for cell type

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12
Q

What are GINAs

A

Genetically encoded indicators of neuronal activity
E.g. GCamp

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13
Q

Advantage of GINAs

A

can be targeted to individual cells and tissues
Can have temporal control

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14
Q

Structure of GCamp

A

a fusion protein
Green fluorescent protein bound to calmodulin and M13

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15
Q

what is FRET

A

flurescence resonance enrgy transfer

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16
Q

what position do molecules need to be for FRET to occur

A

when 2 molecules in close proximity
e.g. when ligand binds to receptor and the wavelength emitted by one will trigger the next to emit its wavelength

17
Q

name of non slective light gated cation channel used in optogenetics

A

channelrhodopsin 2 (ChR2)
responds to blue light
causes influx of cations= depolarisation

18
Q

name of chloride pump powered by light

A

halorhodopsin (NpHR)
responds to yellow light
influx of Cl- = hyperpol

19
Q

what is ChR2 H134R

A

mutated ChR2 channel
his at 134 position to arg
makes channel more conductive

20
Q

what molecule do both channels have built in that detects the light

A

retinal cofactor

21
Q

3 ways to get the channel/pump protein into neurons

A

plasmid ie transfection
transgenic animals
viral vectors

22
Q

what is used to deliver the light in a cell culture vs a whole animal

A

cell culture = jsut normal LED light with collimator
animal = fibre optic probe

23
Q

what is the human, light sensitive GPCR

A

rhodopsin
interacts w/ Transducin (Gt) = decreases cGMP

24
Q

name of technique when optogenetics used to control GPCR pathways

A

OptoXRs

25
Q

how to make chimeric GPCRs that opto can act on in the brain

A

transmemb and extracellular domains of rhodopsin + intracellular domain of any desired GPCR