L12- Using light to study the brain (optogenetics and dyes)

Question 1

What are optogenetics?

Answer 1

Using light to alter ion channel activity

Question 2

What is fluorescence?

Answer 2

Electrons are excited by specific waevlengths of light as molecules absorb photons.
Energy loss occurs quickly- light emitted in longer wavelength

Question 3

How can we measure the movement of Ca when Ca channels open?

Answer 3

With Ca2+ sensitive dyes

Question 4

What is calcium involved in?

Answer 4

1. NT release into synapse via vesicle release proteins
2. singalling cascades
3. cell division
4. blood clotting

Question 5

What are the steps in fluorescence detecting system in calcium imaging?

Answer 5

1. fluorophore- fluorescent molecule (photo bleaching)
2. wavelength filters
3. excitation source (uv light)
4. detector

Question 6

What are 3 fluorescence instruments?

Answer 6

1. Spectrofluoremeter- analyzes fluoerscence (before-after)
2. Flow cytometer- analyzes, sorts, counts microscopic particles in stream of fluid- doesn’t produce image of cell.
3. Fluorescence microscope- filters weak fluorescent light from other wavelength.

Question 7

What is Fura-2?

Answer 7

An ion-sensitive dye

When Ca2+ binds to it, its shape is distorted and it changes colour

Question 8

What’s the problem with indicator dyes like Fura-2? And how do we solve it?

Answer 8

INdicator dyes are polar molceules so can’t cross the membrane.
Add acetoxymethyl ester (AM Ester). Makes it non-polar can cross the membrane. An ester comes off when inside so it can’t recross.

Question 9

What is GCaMP?

Answer 9

A gentically encoded fluorescent calcium indicator.
Can be targetted to individual cells/tissues for spatial control.
Can make a trasngenic animal that expresses the reporter

Question 10

How do you get genetically-encoded indicators into cells?

Answer 10

Transfection- inserting foreign DNA/RNA into cells
methods- 
1. cyoplasmic injection
2. lipid nanoparticles of DNA
3. viral infection
4. Germline incorporation

Question 11

What is Aequorin?

Answer 11

Bioluminescent protein-based Ca2+ reporter,
Can be genetically encoded like GCaMP.
Does NOT need light to be excited
It’s the source of light in jellyfish that excites GFP.

Question 12

What does Guangxitoxin- 1E from the Chinese tarantula do?

Answer 12

Binds to KU2.1 potassium channels.

Fluorescent-tagged chemoselective toxin can show us when specific variants of ion channels are activated.

Question 13

What’s channel rhodopsin?

Answer 13

Rhodopsin from green algae ion channel

Question 14

What’s halorrhodopsin?

Answer 14

Rhodopsin from halobacteria chloride pump

Question 15

What do channel rhodopsin and halorrhodopsin both need?

Answer 15

Retinal cofactor

Question 16

What does directing the expression of optogenic proteins to specific tissues using genetics mean?

Answer 16

Means you can control neurone activation with ligth in restricted cell types

Question 17

What are opsins?

Answer 17

light-senstive proteins across the animal kingdom

Question 18

didn’t understand about drosophila- note to self

Answer 18

.

Question 19

What are microbial opsins?

Answer 19

directly light-gated ion channels

Question 20

What are metazoan animal opsins?

Answer 20

GPCRs (different animals use different G-protein signalling pathways in visual transduction)

Question 21

What are OptoXRs?

Answer 21

Chimeric (fused) proteins made up for:
transmembrane and extracellular domains of vertebrate rhodopsin
intracellular domain of any desired GPCRs