Lecture 3 - methods in modern neuroscience part 2

Question 1

What is Green fluorescent protein (GFP)

Answer 1

protein that emits light and stimulated with a light of a different wavelength, stimulated by blue light and emits green light

Question 2

GFP structure

Answer 2

has beta sheets (barrel arrangement) with an active center that is responsible for the fluorescence

Question 3

what is GCaMP

Answer 3

Fusion of a GFP and two calcium binding proteins (M13 and CAM)

Question 4

how does GCaMP work?

Answer 4

• GFP is modified to not be as fluorescent in normal conditions
• M13 and CAM interact with eachother in presence of calcium
• This interaction causes a conformational change in GFP to become much brighter

Question 5

confocal microscopy

Answer 5

rejects light coming not from the focal plane and decreases area of excitation. this allows massive increase in spatial resolution

Question 6

how does confocal microscopy work?

Answer 6

achieveed by putting a pin hole between the lenses so most of the light that is not in focus will be rejected

Question 7

recording neurons with light

Answer 7

records neurons deeper in tissue and only increases when certain neuron is stimulated

Question 8

problems with recording neurons with light

Answer 8

animal is stressed and sedated using sodium channel blockers. animal does not perform behaviour it usually does

Question 9

virtual reality

Answer 9

• mouse placed on a ball that moves in certain direction when mouse moves. turn of the ball leads to turn of field of view
• objective placed on top as usual to see neuronal activity

Question 10

freely moving mice

Answer 10

• tiny fluorescent microscope placed directly in skull
• image delivered via light guide

Question 11

neuronal stimulation by light: channelrhodopsin

Answer 11

• no selective channel so a lot of calcium rushes into the cell and causes depolarisation
• activated by blue light

Question 12

hyperpolarisation by light : halorhosopsin

Answer 12

• chloride channel activated so potential decreases
• activated by yellow light

Question 13

large scale studies

Answer 13

• function aligned with morphology
• millions of neurons together and study their connections
• creates maps between neurons activated and not activated by light