Dyes & Optogenetics Flashcards
(32 cards)
What is the primary use of reporter dyes in electrophysiology?
To measure neuron activity and ion fluxes in real-time
Reporter dyes help determine spatial distribution and timing of ion movements within neurons.
What is the spatial resolution of electrophysiology?
Local but very high (patch) and global (whole neuron, circuit)
Electrophysiology typically monitors one cell or circuit at a time.
What ions can be measured using calcium-sensitive dyes?
Ca2+, Na+, K+, Cl-
Calcium-sensitive dyes are particularly selective for Ca2+ ions.
What is the role of calcium-sensitive dyes?
They are chelators that bind calcium ions
The binding alters the excitation wavelength, allowing measurement of calcium levels.
Name a type of low-affinity calcium indicator dye.
Fura-2
Used to detect large changes in calcium ion concentration
How are indicator dyes introduced into cells?
- Using Acetoxymethyl Ester (AM Ester)
- Makes FURA-2 non polar as COO- groups are ‘covered’
- FURA-2 AM is non-polar & can’t bind calcium but can cross the cell membrane
- Esterases in the cytoplasm hydrolyse the AM groups
- FURA-2 is now polar and can bind calcium but can’t recross the cell membrane
What happens to Fura-2 after it enters the cell?
It binds Ca2+ but cannot re-cross the cell membrane
Inside the cytoplasm, Fura-2 remains bound to calcium ions.
What are GINAs?
Genetically-encoded Indicators of Neuronal Activity
They allow for targeted measurement of neuronal activity in specific cells or tissues.
What is the function of genetically-encoded indicators like GCaMP?
To report calcium ion levels in real-time in living cells
They can be targeted to specific tissues using promoters.
What is optogenetics?
A technique using light to control neurons that have been genetically modified to express light-sensitive ion channels
It allows precise control over neuronal activity using light.
Name a light-sensitive channel used in optogenetics.
Channelrhodopsin-2 (ChR2)
ChR2 is activated by blue light (λ 470 nm) and allows cation influx.
What is the effect of ChR2 activation on a neuron?
Depolarization (Excitation)
This leads to increased neuronal firing and activity.
What is the role of halorhodopsin (NpHR) in optogenetics?
It acts as a chloride pump powered by light
Responds to yellow/orange light at 590 nm
NpHR is used to induce hyperpolarization (Inhibition) in neurons.
What are OptoXRs?
Chimeric proteins that link rhodopsin domains with GPCR intracellular domains
They enable control of GPCR pathways using light.
What is the significance of using light-responsive GPCRs?
To control intracellular signaling pathways with light
This could enhance the precision of neuromodulation in research and therapy.
Fill in the blank: Calcium-sensitive dyes are used to measure changes in _______.
Ca2+
These dyes indicate the concentration of calcium ions, which are crucial for various cellular processes.
What is an absorption spectrum?
Frequency/wavelength of light that the fluorophore absorbs
What is an emission spectrum?
The light which is emitted when the fluorescence occurs
Principles of fluorescence
Electron becomes excited by UV light
As electron loses energy, it emits a lower energy light
Advantage of using reporter dyes
Can tell where within a neuron, across the whole neuron and when in multiple neurons simultaneously
How are Fura dyes used to measure the concentration of Ca2+ ?
- Excite at 340 nm (has Ca2+ bound) and then again at 380 nm (No Ca2+ bound)
- Measure the emission at 510 nm for each
- Take the ratio:
Measure of calcium at emission from 340 excitation
————————————————————————
Measure of calcium at emission from 380 excitation
Why are dyes so important?
Look at ion fluxes in real time
Determine spatial distribution of ion flux
What is GCaMP?
A fusion protein
Genetically encoded Ca2+ indicators
Can be targeted to individual cells and tissues
Can have temporal control
How can GCaMP be used?
Make a ‘transgenic’ animal that expresses the reporter e.g. CRISPR
Target to tissue/cell specific promoter
Indicator will only be expressed in target cell
