Exam 2 Brainbow

Question 1

What problem does the cre lox system solve?

Answer 1

It solves the problem of the promoter and enhancer of not adequately expressing your protein. Therefore, though you are specifically expressing the protein in a subset of your cells, you don’t get a high level of expression. Cre lox system is useful to express high levels of YGI in cells.

Question 2

How does deletion and inversion work in the cre/loxp system?

Answer 2

• Deletion: two lox sites are in the same orientation and on either side of the DNA. When cre is expressed, it excises that chunk of DNA.
• Inversion: Lox sites are opposing. Cre inverts the DNA into its correct orientation and is able to be expressed.

Question 3

How do indicator and driver mice work?

Answer 3

• The indicator mouse has the indicator gene that you want to express.
• The driver mouse has the gene for cre expressed.
• Combination of the two gets the cre expressed, and deletion of the stop codon between the two same-oriented lox sites.
• Now, the promoters and enhancers are free to express YGI.

Question 4

How do you make sure that the cre is specifically expressed in your subset of cells that you want to study?

Answer 4

You combine the animal with mice that express cre in a certain subset of cells so if you want to study cholinergic cells, you should use cre that is only expressed in cholinergic neurons.

Question 5

Describe cre/lox system with the stop codon and identify a problem with this set-up. Name the method that solves this.

Answer 5

You can design your knockin DNA such that YGI is behind a stop codon. The stop codon is flanked by two same-oriented lox sites. When cre is expressed, then cre identifies those lox sites and removes the stop codon. Then, the promoters and enhancers are free to transcribe YGI. A problem with this is that the promoter might be strong enough to override the stop codon and express YGI even when the stop codon is still there in the absence of Cre.

Use DIO.

Question 6

How does DIO work?

Answer 6

• You use inversion and at least two different types of lox sites. The DNA of interest is between two oppositely oriented lox sites. When Cre is expressed, the DNA is flipped to its correct orientation. Only now does the promoter express YGI. Slide 2.
• Though there are two different pairs of lox sites, the end result is the same.

Question 7

What types of lox sites does the cre act on so that the recombination is between…?

Answer 7

Identical lox sites.

Question 8

Describe the problem associated with visualizing with a GEVI

Answer 8

• The image on slide 3 shows a very clear image because they had to do thousands of trials, which showed clearly where the depolarization occurs and where the action potential propagates.
• Otherwise one electrode would show lots of noise. Putting the electrodes everywhere would cause recording from cells that didn’t receive the depolarizing input.

Question 9

What is the problem with calcium sensors?

Answer 9

They only detect voltage changes due to action potentials and yet they are slow to detect those voltage changes.

Question 10

Describe the recording the researchers did to assess activity in hippocampal neurons. Why did they use FRET?

Answer 10

They stimulated kainite receptors in the CA3 neurons. They used LFP as well as the GEVI. They saw that the fluorescence from the donor decreases and the fluorescence of the acceptor increases because the energy is transferred to the acceptor. They took a ratio of the increase:decrease which gave a higher signal to noise.

-they used FRET because it shows you what cell you are recording from, which you can’t detect through LFP.

Question 11

Why should the graph of voltage vs fluorescence be linear?

Answer 11

If it is not linear then you can’t detect the change in voltage per unit fluorescence. The same fluorescence change can show a higher voltage change.

Question 12

Why are slow kinetics of voltage sensors a problem?

Answer 12

They might miss an action potential. They are generally slower than electrodes but you want them to be as fast as possible to detect the voltage change.

Question 13

What are GECIs?

Answer 13

They are proteins that change their fluorescence based on changes in calcium.

Question 14

What are the advantages and disadvantages of GECIs?

Answer 14

-Advantages: 1. higher signal to noise ratios so they are more sensitive than voltage sensors. 2. they are located in the cytosol, and diffusion to the cytosol is fast. Therefore bleaching is not an issue because you can make them diffuse fast.

Disadvantages: 1. they are calcium sensors so they buffer calcium and interfere with the change in the cell since calcium is a signaling molecule. 2. it could decrease calcium. 3. only detect action potentials, not depolarizations. 4. unlike GEVIs, they are slow and only detect action potentials.

Question 15

What are the advantages and disadvantages of GECIs?

Answer 15

Advantages: 1. higher signal to noise ratios so they are more sensitive than voltage sensors. 2. they are located in the cytosol, and diffusion to the cytosol is fast. Therefore bleaching is not an issue because you can make them diffuse fast.

Disadvantages: 1. they are calcium sensors so they buffer calcium and interfere with the change in the cell since calcium is a signaling molecule. 2. it could decrease calcium. 3. only detect action potentials, not depolarizations. 4. unlike GEVIs, they are slow and only detect action potentials.

Question 16

What are the advantages and disadvantages of GECIs?

Answer 16

Advantages: 1. higher signal to noise ratios so they are more sensitive than voltage sensors. 2. they are located in the cytosol, and diffusion to the cytosol is fast. Therefore bleaching is not an issue because you can make them diffuse fast.

Disadvantages: 1. they are calcium sensors so they buffer calcium and interfere with the change in the cell since calcium is a signaling molecule. 2. it could decrease calcium. 3. Unlike GEVIs, GECIs only detect action potentials, not depolarizations. Unlike GEVIs, they are slow.

Question 17

What are the advantages and disadvantages of GECIs?

Answer 17

Advantages: 1. higher signal to noise ratios so they are more sensitive than voltage sensors. 2. they are located in the cytosol, and diffusion to the cytosol is fast. Therefore bleaching is not an issue because you can make them diffuse fast.

Disadvantages: 1. they are calcium sensors so they buffer calcium and interfere with the change in the cell since calcium is a signaling molecule. 2. it could decrease calcium. 3. Unlike GEVIs, GECIs only detect action potentials, not depolarizations. Unlike GEVIs, they are slow.

Question 18

What cells innervate the planar dendritic field of the Purkinje cells?

Answer 18

The parallel fibers from the granule cell neurons (which are inhibitory).

Question 19

What is the location and function of the parallel fibers?

Answer 19

They are parallel to the surface of the cerebellum. They function to control the activity of the Purkinje neurons.

Question 20

Describe the experiment where GECI was expressed in granule cell neurons. What was concluded?

Answer 20

They induced expression of GECI in granule cell neurons and stimulated the superficial layer of granule cell axons. They measured the change in fluorescence/baseline fluorescence. The greater the change in fluorescence the warmer the colour. Thus, if you see a greater change in fluorescence, then the granule cell neurons show action potential, which means the GECI works in detecting calcium changes during depolarization.

Question 21

Why can you use GECI in depolarization but not hyperpolarization events?

Answer 21

Calcium levels change in depolarization but do not in hyperpolarization.

Question 22

How did the researchers visualize the oscillations of zebrafish?

Answer 22

They expressed GECI in the motor neurons in the brains of the zebrafish. They saw change in fluorescence/fluorescence alternating. This is good because it shows that the fish is swimming through contractions, which the GECI detects through depolarization (calcium influx) events.

Question 23

How does the un-innervated eye receive inputs if one eye is connected to layer 4 of the cortez?

Answer 23

The un-innervated eye is connected to adjacent neurons in the layer 4, that connects to other neurons in other layers to be able to send visual information from both eyes.

Question 24

How did the researchers conclude that one eye innervates layer 4 neurons?

Answer 24

They injected radioactive proline into one eye. Then monitored the movement of the proline as it got taken up into proteins. They ultimately found the proline input from one eye was in one segment the layer 4.

Question 25

Describe the relationship between bars of light and the layers of the cortex?

Answer 25

Each cortical layer responds differently to bars of light sent from the right or left eye. Layer 1 neurons are only responsive to input from the left eye. As you go deeper into the layers, the response becomes more biased towards the right eye. Layer 7 neurons are only responsive to input from the right eye.

Question 26

Direction of movement is ______to the direction of the bar of light.

Answer 26

Perpendicular.

Question 27

What is known about the orientation map in layer 2/3?

Answer 27

The researchers showed that the innervation corresponds to different orientation sensitivities in the visual cortex. The orientation map shows which neurons in layer 4 are sensitive to certain orientations of light.

Question 28

Does circuity change as a result of exposure to different orientations of light and how do you know?

Answer 28

It slightly changes. When they recorded calcium fluxes with different orientations and different directions, they saw that some got a better response. They then returned to the same neuron later to perform the same experiments. There was a small change and an increase in bias towards the vertical.

Question 29

Why would researchers not make a transgenic animal that would label all of the layer 2/3 neurons to increase sample size?

Answer 29

That would label all of the neurons and you wouldn’t be able to identify your neuron any more.

Question 30

Describe multiphoton/2-photon imaging

Answer 30

When light is concentrated to one thin section of tissue, the light adds such that the wavelength decreases and the energy increases.

Question 31

What are the advantages of multiphoton/2-photon imaging?

Answer 31

• Reduces out of focus contributions.

- long wavelength light is used, which allows you to focus on deeper sections of the tissue.

Question 32

What problem does brainbow solve? How?

Answer 32

• It allows you to distinguish neurons of the same type form each other.
• It expresses 3 fluorophores at different levels so each cells has distinct levels of each fluorophore.

Question 33

What is the advantage of brainbow in looking at the dynamics of axons?

Answer 33

Brainbow labels axons and cell bodies so you can trace different neurons to different target tissues.

Question 34

Describe brainbow using excision cre/lox system

Answer 34

slide 12

Question 35

Describe brainbow using inversion cre/lox system

Answer 35

slide 13

Question 36

Describe brainbow using a combination of inversion and excision cre/lox system

Answer 36

slide 14

Question 37

What is the problem with brainbow labeling? How do you solve this?

Answer 37

You will have a default colour (red, blue, green) when you don’t have recombination. Thus, even after you have recombination via cre/lox system, the default colour will persist because the cell doesn’t have a way to destroy the proteins that give off that colour. So that limits you to the colours you can have.

-Make the system such that without cre, you won’t have any colour, even a default colour.

Question 38

How do you get varieties of colours using brainbow?

Answer 38

You combine the constructs so the cre acts randomly on each one, and you will get combinations.

Question 39

What is the solution to not having any way to degrade the default colour?

Answer 39

Come up with combinations of constructs for the Cre to randomly act on.

Question 40

How do you fix tissue?

Answer 40

Formaldehyde is divalent so it can cross link two proteins together. These are covalent bonds and everything in the cell is cross linked together and frozen.

Question 41

What is the problem of having fixed tissue?

Answer 41

All the proteins are crosslinked covalently and dead. Therefore you would get less fluorescence.

Question 42

How do you use antibodies to identify the fluorescent proteins to solve the problem of fixed tissue?

Answer 42

1. The antibody identifies the fluorescent protein
2. the fluorescent antibody binds to another antibody
3. you detect that antibody which improves the signal:noise

Question 43

What is the problem with using antibodies to identify fluorescent proteins and what is the solution?

Answer 43

Problem: all of the fluorescent proteins are derived from GFP so the antibody would cross react with other fluorescent proteins.

Solution 1: use tags that are antigenic. The tags bind to the fluorescent proteins and the antibodies bind to the tags. Therefore, you don’t get cross reactivity because the tags are antigenic.

Solution 2: Use less fluorescent proteins. If you use GFP and RFP, they are evolutionarily different so their sequences don’t show overlap and there would be no cross-reactivity with the antibodies.