Methods in Modern Neuroscience

Question 1

What are Sharp Electrode Recordings used to measure?

Answer 1

Membrane potential changes, resistance, time constant, synaptic potentials and action potentials of a whole neuron.

Question 2

How is a Sharp Electrode Recording peformed?

Answer 2

Insert the electrode into the cell body (soma) of a neuron or a thick dendrite.

Question 3

What are 3 disadvantages of sharp electrode recordings?

Answer 3

1) Cannot change the solutions inside or outside the cell (some experiments require this).

2) Can’t control the membrane potential as when the electrode stabs the membrane their connection is not tight so ions can flow between the glass and the membrane.

3) Cannot measure single channels (Patch clamp can be used for this).

Question 4

What are 5 model organisms used in Neuroscience?

Answer 4

1) Humans

2) Primates

3) Lower mammals (cats but mainly rodents)

4) Lower vertebrates (zebrafish)

5) Invertebrates (Drosophila)

Question 5

What are the 4 steps of a methodological approach to study a new neuronal type?

Answer 5

1. Describe morphology of this part of the brain
2. Map connections of neurons in this part
3. Describe activity
4. Theoretical study

Question 6

What are 2 methods for a electrophysiological recording of neurons?

Answer 6

Sharp electrode recording and Patch clamp recording

Question 7

What are the 5 differences between a Patch Clamp Recording and a Sharp Electrode Recording?

Answer 7

1) Patch clamp uses an electrode with a larger hole.

2) Patch clamp sucks up apart of the membrane almost like a pinch, whereas the sharp electrode stabs a hole in the membrane.

3) Patch clamp can control membrane potential, Sharp electrode cannot.

4) Patch clamp can label individual neurons with dye (can characterize morphology as well as electrophysiology) while the sharp electrode can only measure electrophysiology.

5) Sharp electrode recording measures potential of whole neuron while Patch clamp can for an individual ion channel.

Question 8

What are the names of the 4 pipette based Patch Clamp types?

Answer 8

1) On-cell (cell attached)

2) Inside-out

3) Whole-cell

4) Outside-out

Question 9

Why can a Patch clamp control the membrane potential of a neuron but a Sharp electrode recording cannot?

Answer 9

Patch clamp creates a tight connection between the membrane and electrode (as engulfs parts of the cell into the electrode) so movement of ions is controlled instead due to lack of contact with extra cellular fluid. Sharp electrode stabs a hole so ions moves out and in of cell.

Question 10

How is an On-cell (cell attached) Patch Clamp done and what does it measure?

Answer 10

> The electrode is brought close to the membrane, touch the membrane and apply negative pressure which sucks a part of the membrane into the electrode (membrane stays intact)

> Measures activity of individual channels on the part of the membrane sucked into the electrode.

Question 11

How is an inside-out Patch Clamp done and what does it measure?

Answer 11

> While in the cell-attached patch configuration, remove electrode and the membrane that is stuck in the electrode (the patch) is pulled away from the cell membrane.

> This exposes the cytosolic surface of the membrane to the controlled solution on the outside, so we can measure activity of channels on intracellular side in a controlled solution.

Question 12

How is a whole-cell Patch Clamp done and what does it measure?

Answer 12

> While in the cell-attached patch configuration, can apply stronger negative pressure which breaks part of the membrane, so the solution within the pipette/electrode is in contact with the cell’s cytosol.

> So it can control the membrane potential, record currents and the membrane potential of the whole cell.

Question 13

How is a outside-out Patch Clamp done and what does it measure?

Answer 13

Whole-cell method applied and then retract pipette after the rupture. Causes part of the membrane to detach and form a small vesicular structure in the pipette. This is in contact with the external solution so can be used to measure activity of a single channel while the intracellular side is in contact with the extra cellular fluid.

Question 14

How is a current voltage relationship of voltage gated ion channels measured?

Answer 14

> Single channel recordings so are done by: On-cell, Inside-out, Outside-out patch clamps (not whole cell)

> Alter the membrane potential of the cell and measure the activity of the channel, keep doing this at a different membrane potential to create a mV curve on a graph.

Question 15

How can a patch clamp be used to characterise the morphology of a neuron?

Answer 15

An individual neuron can be labelled by having a fluorescent dye (usually Lucifer Yellow) inside the electrode, the dye diffuses from the electrode into the cell and travels through the whole neuron.

Question 16

What are 4 issues with the patch clamp technique?

Answer 16

1) Cannot label many cells
>As pipette can only clamp to one cell at a time, only 1 cell can be dyed per experiment.

2) Limited ability to label specific cell type
>If the area is 50/50 filled with 2 types of neurons, we only have a 50% chance of labelling the neuron interested in.

3) Limited ability to label cellular compartments
>We can only label the whole cell, not the compartments

4) Limited ability for live labelling
>Fluorescent dye usually effects the neuron, so the behaviour is different from an intact neuron.

Question 17

What does GFP stand for?

Answer 17

Green Fluorescent Protein

Question 18

What is the structure of GFP?

Answer 18

Active centre responsible for fluorescence, surrounded by beta sheets.

Question 19

How does a) Excitation b) Emission work for GFP?

Answer 19

If stimulated by blue light (430nm), the active core emits green light (510nm).

Question 20

What is GFP used for?

Answer 20

Live labelling of individual neurons

Question 21

What 2 factors differ between different fluorescent proteins?

Answer 21

1) These proteins have different excitation wave lengths (light that shines on protein to cause emission)

2) Different emission wavelengths (light that reflects from protein)

Question 22

What is the wavelengths of excitation and the colours of emission for a) ECFP b)EYFP c)DsRed fluorescent proteins? (Don’t think i need to know)

Answer 22

a) ECFP
>Excited by 430-440nm (dark blue light)
>Emits light blue light

b) EYFP
>Excited by 488nm (light bluish light)
>Emits light green light

c) DsRed
>Excited by 550nm (yellow light)
>Emits red light

Question 23

How could we use fluorescent proteins to label different cell types?

Answer 23

Can express different fluorescent proteins in different cell types, so can label different cells with different colours

Question 24

How can fluorescent proteins be used to label organelles?

Answer 24

Can do fusion of one fluorescent protein with a protein localized in different organelles.

Question 25

What are the three needed parts to make a fluorescent microscope?

Answer 25

Emission filter, Excitation filter, and a Dichroic mirror.

Question 26

What is the function of a) Emission filter b) Excitation filter c) Dichroic mirror in a fluorescent microscope?

Answer 26

a) Excitation filter, causes only the wanted wave length of light used to stimulate the sample/ exit via the objective lens (more precise wave length)

b) Emission filter, allows us to only pass the wave length of light we want through the tube lens (to the eye)

c) Dichroic mirror, reflects light with certain wavelength to the objective lens and passes a wavelength through with a particular wavelength to the tube lens.

Question 27

How would a fluorescent microscope be set up for GFP?

Answer 27

for EGFP (green protein) we would need a blue excitation filter to allow blue light out the objective lens to stimulate GFP, a green emission filter that reflects other light but passes green light into the tube lens and a dichroic mirror which passes green light to tube lens (for eyes) but reflects blue light to objective length (to sample)

Question 28

What would be the effect of removing a dichroic mirror from a fluorescent microscope?

Answer 28

If dichroic mirror was removed, a lot of blue light would come onto the tube lens so we would have a string background.

Question 29

What are 2 reasons GFP is used?

Answer 29

GFP can be used to understand the morphology and the function of neurons.

Question 30

What is GCaMP made of?

Answer 30

GFP fused to M13 and Calmodulin proteins (calcium indicators)

Question 31

What happens to GCaMP in the presence of calcium?

Answer 31

M13 and Calmodulin proteins interact which changes the composition of GFP making it become brighter (fluoresces green).

Question 32

When a neuron is active, what happens to GCaMP and why?

Answer 32

When a neuron is active Ca2+ is present which causes GFP to fluoresce green.

Question 33

Why are zebrafish useful for imaging the brain?

Answer 33

Zebra fish have a small brain and are transparent so we can image their whole brain showing the activity of the neurons.

Question 34

What is Confocal microscopy?

Answer 34

Confocal microscopy rejects light coming not from the focal plane and decreases the area of excitation (only collect light that is emitted from the place that is in focus currently).

Question 35

How is Confocal microscopy carried out?

Answer 35

Put a small pin hole between the lenses where they focus on (few microns), then will only collect the light from the part of the sample in focus.

Question 36

What is advantageous about using Confocal microscopy over wide field microscopy when studying neuron activity?

Answer 36

Confocal microscopy has much increased spatial resolution than wide field microscopy

Question 37

What is a two-photon microscope and what is its advantage?

Answer 37

It is a modified confocal microscope that is able to image fluorescence deeper in the tissue as it uses infra-red light.

Question 38

What are we able to observe if we use confocal microscopy with GCaMP?

Answer 38

Using confocal microscopy with GCaMP we can correlate the sensory stimulation of the nervous system with the activity of individual neurons.

Question 39

What are 3 issues with using confocal microscopy with GCaMP on animals?

Answer 39

1) Animal is sedated using Na+ channel blockers
>Sedated as if animal moves, we image from a different part of the brain, but blocking Na channels effects the excitability of the neurons we study.

2) Animal is stressed
>Stress releases neuro modulators which effects neurons we are studying.

3) Animal does not perform behaviour that it usually does
>As they are sedated, we can’t measure their neuron activity while they are doing certain behaviour such as hunting.

Question 40

What are 2 solutions to using sedation when using GCaMP to measure neuron activity?

Answer 40

1) Virtual reality surrounding animal on a ball that spins but stays stationary when they run.

2) Freely moving animal with tiny fluorescent microscope placed on skull

Question 41

What is an advantage and a disadvantage of using virtual reality for studying neuron activity with GCaMP?

Answer 41

> Can record activity of individual neurons during active behaviours such as running from a predator.

> It is hard to do with fish.

Question 42

What is an advantage and a disadvantage of using freely moving mice for studying neuron activity with GCaMP?

Answer 42

> Can record activity of individual neurons during active behaviour of mice.

> Lower quality than with a larger microscope.

Question 43

What is an advantage and a disadvantage of using a microscope which follows a moving fish for studying neuron activity with GCaMP?

Answer 43

> Records individual neuron activity in freely active fish.

> Images are not great quality

Question 44

Instead of virtual reality, how can we measure individual neuronal activity in fish with GCaMP?

Answer 44

Use a microscope paired with a camera that detects the movement of the fish, the model predicts where the fish will move next and the microscope moves in advance to record the fish neurons.

Question 45

What is a method used to activate neurons without use of electrodes?

Answer 45

By applying light to neurons containing Channelrhodospsin which will open allowing Na+ ions to enter and fire action potentials.

Question 46

What is Channelrhodospsin and how is it effected by light?

Answer 46

> A non-selective ion channel

> When applied with blue light (488mm) it opens and Na+ ions enter the neurons which causes depolarisation.

Question 47

What is Halorhodopsin and how is it effected by light?

Answer 47

> A chloride channel

> When yellow light (570nm) is applied, it opens and chloride rushes into the neuron, hyperpolarising it.

Question 48

What is the effect of shining a) blue light (488nm) b) yellow light (570nm) onto a cell containing Channelrhodospsin and Halorhodopsin?

Answer 48

a) When applying blue light it starts spiking due to depolarisation by Na+ entering

b) When applying yellow light it stops the APs due to repolarisation by Cl- entering

Question 49

How can we test the function of a neuron in a process without using electrodes?

Answer 49

If we use apply yellow light on its own and Halorhodopsin is present, the neuron will stop activity and we can see the effect on the overall process.

Question 50

What is a connectome and what is it used for?

Answer 50

A map of all the connections of a neuron, and it allows us to reconstruct the function of these individual neurons.