Methods In Neuroscience 2 Flashcards

1
Q

What is the key principle of a fluorescent microscope?

A

Blue light is shone at a GFP through a microscope. The GFP then emits a green light which is detected..

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2
Q

In detail how is blue light shone at the GFP via the fluorescent microscrope?

A

Blue light is first turned on.

It then passes through an excitation filter (which blocks other wavelengths of light getting through)

This blue light is then reflected off a dichroic mirror and shone through an objective lens onto the GFP

Excitation filter is on exam

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3
Q

How is green light detected from GFP?

A

Green light comes off the GFP and goes back through the microscopes objective lens

This then passes up the microscope and goes THROUGH the dichroic mirror (it isnt reflected)

The light then passes up through an emissions filter which doesnt let any other light through

This is then detected and seen through a tube lens

Emissions filter is on exam

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4
Q

What are GCaMP - GFP based calcium indicators?

A

These are proteins based on GFP which help us to detect calcium

An example = cpEGFP

In this example there are two proteins bounds M13 and CaM

When these proteins bind to calcium in solution the GFP shine

This can help us detect calcium in the brain, especially when neurones become active.

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5
Q

The higher the resolution of a microscope, the better

Which is better a wide field or confocal microscope?

One exam!

A

Wide field collects light from below and above the focal plane. This is saying it collects light outside of the focal plane. Whereas confocal only uses light from the focal plane
This reduces their area of excitation
To excite the whole sample you use a laser to scan all parts of the sample.

However:
Confocal microscopy rejects light not coming from the focal plane and decreases the area of excitation.
This produces images at a much better resolution than the wide field microscope

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6
Q

Problems with using GFP microscopy to study the brain

A

The animal is usually sedated using sodium channel blockers

Thus the animal becomes stressed

Also these channel blockers make neurones less excitable. The animals neurones dont perform usual behaviours..

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7
Q

Solutions to the disadvantages of GFP microscopes? (Problems of sedating animals)

A

You could not sedate the animal

Make the animal believe it is acting in a certain way i.e. via virtual reality

Or using a free moving animal

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8
Q

How to use virtual reality to study the brain?

A

Do with mice
Put animal on a bowl that moves in different directions . Allow animals to turn
Keep head of animal fixed
Usually train animal to do this voluntarily

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9
Q

Free moving methods of testing an animals neurones?

A

Fit a tiny fluorescent microscope to an animal like a mouses head
A image can be made by a light guide . I.e. a light is shone which actives neurones in the brain

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10
Q

How to do neuronal stimulation by light? Using channelrhodopsin.

A

Either stimulate a neurone and observe the behaviour

Or inhibit a neurone and then see if there is an absence of behaviour

You can use blockers in the brain which can excite or inhibit neurones
You can also induce mutations which can make neurones become stimulated more or less

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11
Q

One example of using channel rhodopsin to study neuronal stimulation by light

A

Insert artificial channels into an organisms brain which respond to light

This can be a sodium potassium channel and doesnt have to be extremely selective

This can open the neurone

This will lead to the membrane being polarised.

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12
Q

How can we use Halorhodopsin = chloride channel to study neuronal stimulation by light?

A

This halorhodpsin channel usually hyperpolarises the membrane

We can study this by shining a light and waiting for activation

This channel helps us to understand certain conditions, like epilepsy.

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