Module 13 - Optogenetics, dyes and radioligands

Question 1

Dyes

Answer 1

A substance that binds to something else and has its optical activity changed

Question 2

Fluorescent

Answer 2

Calcium fluoride and fluorite are the basis of its name due to them absorbing UV light and re-emitting it at a shorter wavelength

Question 3

Fluorophore

Answer 3

Anything that exhibits fluorescence

Question 4

Why do we use reporter dyes

Answer 4

Allows for the when, what, and where in neurones to be monitored without being confined to damaging cells or cells being too small (ie with electrophysiology, dendrites are too small for clamping)

Question 5

FURA-2: what is it and how does it enter cells?

Answer 5

A calcium dye which has to four carboxylic acid groups that attract calcium

FURA-2 cannot enter cells due to it being polar but FURA-2AM (acetoxymethyl) can cross due to the carboxylic acid groups being esterified. Once in the cell, FURA-2AM is cleaved by esterases and it can attract calciums again

Question 6

GINAs: what are they, what are some of their major strengths, and what is one example?

Answer 6

Genetically encoded indicators of neuronal activity

Can be targeted to individual tissues/cells, has temporal control - can choose when the indicator is active

GCamP

Question 7

GCaMP

Answer 7

Green fluorescent protein + calmodulin + M13

By inserting it into the genome of an animal just in front of the promoter, it can be produced and expressed in an exact cell type/tissue

Question 8

FRETs

Answer 8

Fluorescence resonance energy transfers

Contain two fluorophores (acceptor/donor) which has the donor fluorophore as fluorescent when interacting with light alone, but emits the acceptors light when they are within close proximity as energy is transferred

Question 9

Light-activated proteins: animals and microorganisms

Answer 9

Animals - Opsins (GPCRs)

Chlamydomonas - Rhodopsin (7 trans-membrane protein with a peak absorption at ~417nm (blue light). The protein is a non-selective ligand-gated cation channel that is similar to GPCRs)

Halobacteria - Halorhodopsin (7 transmembrane domain protein that is a chloride pump that absorbs light at 590nm (orange/yellow light))

Halobacteria - bacteriorhodopsin (uses light as a protein pump)

Question 10

ChR2

Answer 10

Channel-rhodopsin 2:

Built-in retina molecule which detects light and, in the presence of blue light, opens the ion channel

Question 11

ChR2 and NpHR and their possible effects on optogenetics

Answer 11

Channelrhodopsin-2 could be used to produce an excitatory effect as activation of the retina molecule would cause an influx of positive ions (mainly sodium ions), causing depolarisation

Halorhodopsin could be used to produce inhibitory effects as activation of the molecule would cause an influx of chloride ions, causing hyperpolarisation

Question 12

Introducing ChR2/NpHR into animals: the process behind adding the gene, and activating the gene

Answer 12

Transfection:

The plasmid containing the gene is put in a viral vector and inserted into the animal’s hippocampus and then the channel is activated by fibre-optic probes which shine blue light on the channels

Question 13

OptoXRs: what are they and what possible opportunities can they bring?

Answer 13

Chimeric proteins containing the 7 transmembrane domain of animal rhodopsin and the intracellular domain of any desired GPCR