modern methods 1+2

Question 1

lesion experiments

Answer 1

where whole parts of the brain are removed, and compare behavioural output in control animals and animals with lesions

Question 2

2 types of lesions

Answer 2

directed - done on model animals

naturally occurring - after strokes and other neurodegenerative diseases

Question 3

whole brain imaging techniques

Answer 3

fMRI, non invasive used in visual processing studies

Question 4

methodological approach to study a new neuronal type

Answer 4

first - cluster neutrons into different types by describing their morphology
second - map connections of neurons
describe activity

Question 5

what does a sharp electrode recording measure

Answer 5

measures the electrophysiology of a cell

Question 6

what is a sharp electrode

Answer 6

an electrode with a very thin tip filled with electrolyte
inject the current into the cell and record the changes In membrane potential and can stimulate and calculate the number of spikes

Question 7

how do you know if the electrode is not inside the neuron

Answer 7

will measure a potential of 0mv

Question 8

disadvantages of sharp electrode recordings

Answer 8

no solution change inside or outside of the cell
limited possibility for controlling the cell membrane
cannot measure single channels - as measure the potentials generated by the entire membrane

Question 9

what is the patch clamp method

Answer 9

electrode has a larger tip diameter. use glass electrode filled with electrolyte similar to intra(KCL) or extra(NaCl) solution.
touch the cell and apply negative pressure. part of the membrane will form a tight junction with the electrode and will be able to record the currents of individual channels

Question 10

what is an inside out patch clamp ?

Answer 10

pull part of the membrane

Question 11

what is a whole cell configuration patch clamp ?

Answer 11

brake the membrane and record currents of the whole cell

Question 12

what is an outside out configuration

Answer 12

when in a whole cell configuration, pull the membrane giving and outside-out configuration

Question 13

what is the difference between inside out and outside out configurations?

Answer 13

inside out - can change the intracellular solution and see the cytoplasmic domain of the specified channel

outside out - can change the extracellular solution, and see how this affects outer part of the channel

Question 14

how can we measure morphology and physiology in one experiment

Answer 14

making a whole cell and add fluorescent dye in the pipette solution

Question 15

why will the dye only label one neuron

Answer 15

as not soluble in water so can’t spread unless there are gap junctions

Question 16

problems with dye and labelling

Answer 16

cannot label many cells
limited ability to label specific cell type
limited ability to label cellular compartments
limited ability for live labelling

Question 17

what can be used instead of dye

Answer 17

Green fluorescent protein (GFP)

Question 18

what is GFP

Answer 18

fluorescent protein
stimulated by blue light
emits green light

Question 19

wavelength of blue light

Answer 19

450-495 nm

Question 20

wavelength

Answer 20

495-570nm

Question 21

what do you need to image GFP

Answer 21

a fluorescent microscope

Question 22

what are the important modules of a simple fluorescent microscope

Answer 22

objective lens and tube lens
eye/camera at the top
form of excitation light (blue)
excitation filter - rejects anything that isn’t blue light
emission filter - so for GFP rejects everything not green
dichroic mirror - passes light with a certain wavelength and rejects light with another wavelength

Question 23

how can GFP be used to understand the function o a neuron

Answer 23

live imaging
genetically encoded calcium indicators - proteins based on GFP, stabilised in a dim conformation
two different proteins - calmodulin and M13

Question 24

functions of calmodulin and M13 in GFP imaging

Answer 24

interact with each other in presence of calcium causing entire molecular to be stablished in a bright conformation
can observe activity of calcium - neuron active = calcium levels rise and neuron becomes brighter

Question 25

what is wide field microscopy

Answer 25

collects light from below and above the focal plane

Question 26

what is confocal microscopy

Answer 26

rejects light not coming from focal plane and decreases the area of excitation

Question 27

what is infa-red light used for

Answer 27

can go deeper into the tissue than blue light and allows to increase amount of tissue observed

Question 28

problems of high resolution microscopy on animals

Answer 28

animal sedated using Na+ channel blockers which decrease excitability of many neurons
animal is stressed
animal does not perform normal behaviour

Question 29

solutions of problems in high resolution microscopy

Answer 29

virtual reality

freely moving animal

Question 30

what is channelrhodopsin

Answer 30

non selective channel stimulated by blue light, depolarises the neuron

Question 31

what is halorhodopsin

Answer 31

chloride channel activated when stimulated with yellow light
leads to hyper polarisation of the membrane

Question 32

benefits of confocal microscopy

Answer 32

can control depth of field, elimination or reduction of background information away from the focal plane