modern methods in neuroscience

Question 1

State the mechanism of Sharp electrodes

Answer 1

• Sharp electrode with a thin tip filled with an electrolyte such as potassium chloride
• Inject a current into the cell and record the changes in membrane potential
• Look at the action potentials

Question 2

What are the disadvantages of sharp electrode recordings?

Answer 2

• No solution change inside or outside the cell (some electroinjection is still possible)
• Limited possibility for controlling membrane potential
• Cannot measure single channels
• Can disrupt neuron
• Can’t measure single channels as measuring current and potential of entire membrane

Question 3

When might Sharp electrodes be used instead of patch clamp?

Answer 3

to study isolated cells or in intact tissue

much smaller pore so little ion exchange between elected and ICF

Question 4

Describe microelectrodes with example using Drosophila

Answer 4

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in livingDrosophila.

e.g. record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Question 5

What is patch clamp?

Answer 5

A method that enables an investigator to hold constant the membrane potential of a patch of membrane while current through membrane channels is measured.

Question 6

What are the four patch clamp methods?

Answer 6

On-cell, whole-cell
inside-out
Outside-out

Question 7

Describe On-cell patch clamp

Answer 7

Go closer to cell, apply gentle suction – connection between electrode and membrane
Use glass electrode filled with electrolyte.

Question 8

Describe inside out patch clamp

Answer 8

pull the part of membrane, patch is remove – can change ICF solution

Question 9

Describe Whole-cell patch clamp

Answer 9

break the membrane by applying stronger suction, so can read the ICF solution for whole cell

Question 10

Describe outside-out patch clamp

Answer 10

break membrane and then pull out – can change ECF solution and isolate the channels

Question 11

What’s the difference between inside/outside and whole cell?

Answer 11

Inside and outside measure individual channels

Whole cell- measure whole cell

Question 12

how do you study Morphology and electrophysiology in one experiment

Answer 12

Adding a fluorescent dye in the pipette solution will give information about the morphology of the recorded neuron
Can combine morphology and electrophysiology
Doesn’t go through membrane – dye will slowly diffuse into cell
Only label one neuron unless there are gap junctions

Question 13

What is electrophysiology?

Answer 13

Electrophysiologyis the study of ionic currents and electrical activity in cells and tissues

Question 14

problems with using adding fluorescent dye to study morphology

Answer 14

Problems
- Cannot label many cells
- Limited ability to label specific cell type
- Limited ability to label cellular compartments
- Limited ability for live labelling
Patch clamping mainly done in slices??

Question 15

Describe Green fluorescent protein

Answer 15

• Fluorescent protein
• Stimulated by blue light
• Emits green light
• Contains a centre that allows protein to emit green light if stimulated by a blue light