Exam 2 Circuit Analysis 1

Question 1

What is circuit analysis?

Answer 1

Assessing the connections and activity of the nervous system.

Question 2

What is EEG? How is it performed? What does it show? What are the advantages and disadvantages?

Answer 2

• EEG records the depolarization of thousands of neurons under an electrode.
• Advantage is that it has good temporal resolution, and see more deeper into the gyri. Another advantage is that you can do it over the entire surface of the brain.
• A disadvantage is that it has poor spatial resolution because it has cm depth.

Question 3

How does the EEG reading differ based on how coordinated the depolarization is?

Answer 3

• Coordinated depolarization shows smooth waves that have a high amplitude and low frequency. -
• Uncoordinated depolarization just shows noise, with low amplitude and high frequency waves.

Question 4

What is MEG, how is it better than EEG and how is it worse than EEG?

Answer 4

• MEG uses an electric field and a magnetic field to record activity.
• Better: has better spatial resolution than EEG (mm resolution). It is magnetic, so the skull and other tissue between the magnet and neurons don’t interfere with the readings.
• Worse: doesn’t give as much depth, so you are left with looking at the superficial levels, and can’t see into the gyri, which EEG lets you do.

Question 5

What is local field potential, what are its advantages, what are its disadvantages compared to the other imaging techniques?

Answer 5

• Local Field Potential lets you look at action potentials from a small group of neurons measure with one electrode.
• If you use multiple electrodes you can identify which neurons are undergo action potentials. You can’t study action potentials in EEG or MEG.
• It has better spatial and temporal resolution than MEG or EEG.
• A disadvantage is unlike EEG, you can’t do it over the entire surface of the brain.

Question 6

What is EcoG?

Answer 6

It is similar to EEG. You insert the electrode beneath the skull so it covers a surface of the brain.

Question 7

What are the differences in resolution for EEG and LFP?

Answer 7

EEG has a lower frequency resolution because you cover a greater part of the cortex. LFP has higher frequency resolution over a small area of the cortex.

Question 8

What are optogenetic sensors and how do they work?

Answer 8

They are proteins. You put the promoter and enhancer in front of the pseudogene that encodes the fluorescent protein.

• The fluorescent protein is specific to the neuron that you are recording from so it fluoresces in response to changes in that neuron.
• They allow you to sense a change in the neuron’s activity by sensing a change in voltage or calcium in the event of an action potential voltage.

Question 9

What are the advantages of using optogenetic sensors over using other approaches?

Answer 9

• The optogenetic sensors are permanently expressed since the changes are genetic. Thus, you can image for long periods of time or return to the same cell later.
• This method is less invasive since you are not introducing electrodes into the animal. Other approaches limit you to how long the animal stays alive for.
• Optogenetic sensors allows you to identify the neuron that you are recording from.

Question 10

Why do scientists change the amino acid sequences of GFP and RFP?

Answer 10

Changing the amino acid sequences enhances the fluorescence of the natural colours. They are helpful because the tissues don’t interfere with the longer wavelength dyes as much so you can see more deeper into the tissue than you would had you used short wavelength dyes.

Question 11

Function of aequorin?

Answer 11

As calcium levels increase, there is a chemical reaction that occurs inside aequorin that makes it produce CO2 and fluores blue light.

Question 12

How does fluorescence normally work?

Answer 12

An electron absorbs light energy and is excited to a higher energy orbital. It releases a lower energy, higher wavelength light as it falls back to its ground state.

Question 13

What is FRET and how does FRET work at the level of the electron?

Answer 13

• FRET is a type of resonance energy transfer.
• The first electron absorbs light energy and is excited to a higher energy orbital. It falls to the ground state and releases lightless energy, which is absorbed by a second neighbouring electron.
• The neighbouring electron is excited to a higher energy orbital. When that electron returns to the ground state, it releases the energy as light.
• We never see light released by the donor, perhaps it might be faint.

Question 14

How does FRET work when you couple it with a GEVI?

Answer 14

Under resting conditions, the donor and acceptor are too far away from each other so FRET does not take place. A voltage reversal snaps the donor and acceptor into close proximity to one another. Then you periodically excite the donor so it transfers the energy to the acceptor. Then the acceptor will give off the light energy. The light that you see is from the donor.

Question 15

What happens if the donor and acceptor are not close to each other and you excite the donor with fluorescent light?

Answer 15

The donor will fluores its colour and you don’t get FRET.

Question 16

What is an advantage of taking the ratio of increased fluorescence from GFP to decreased fluorescence of blue fluorescent protein?

Answer 16

That enhances the signal, and controls for variability between the cells so the ratio isn’t affected by external factors such as thickness of cell. It also minimizes signals from random noise.

Question 17

What is the function of optogenetic actuators?

Answer 17

They are proteins that alter the depolarization or hyperpolarization (excitation or inhibition) of cells. Thus, they alter the action potential of the cell.

Question 18

What is channelrhodopsin and why is it excitatory?

Answer 18

Channelrhodopsin is an optogenetic actuator. It is excitatory because potassium has less driving force into the cell since the resting membrane potential of the cell is close to the equilibrium potential of the potassium. Sodium and calcium have a large driving force into the cell so the cell undergoes depolarization.

Question 19

What are the two inhibitory optogenetic actuators and what are their functions?

Answer 19

Arc pumps: They are optogenetic actuators that hyperpolarize the cell by pumping protons out.

Halorhodopsin: It is an optogenetic actuator that pumps Cl- into the cell and hyperpolarizes it.

Question 20

Why is it convenient that ChR2 and NpHR respond to different wavelengths of light?

Answer 20

You can put them in the same cell and couple them. Thus, you can use one wavelength for the ChR2, and another wavelength for the NpHR and cause both excitatory and inhibitory responses in the same cell.

Question 21

How do you introduce GEI’s into cells via in utero electroporation? What is a disadvantage and how can this also be an advantage?

Answer 21

Inject plasmid that has the promoter and enhancer for gene expression in cells that you want gene expression into tissue. Then you use electroporation to let the plasmid DNA enter the cells. A disadvantage is that you don’t label all of the cells with the plasmid. This can be an advantage because you can use the cells that don’t have the plasmid as a control to see how the transfection influenced the neurons. Another advantage is that you don’t have to grow transgenic mice and you can do the experiments right away.

Question 22

How do you make targeted injection of plasmid DNA?

Answer 22

You use neuron birth dates. The later born neurons are in the more superficial layer of the neocortex since they travel outward. Thus, the later in development you do the injection, the more superficially the plasmid will be.

Question 23

Where does stem cell division take place and what is the result of the divison?

Answer 23

It takes place at the ventricular surface and gives rise to neurons.

Question 24

How to make a viral vector and how is it injected into cells?

Answer 24

You grow the virus in cells. Then pressure inject the virus into the brain. The receptors on the cell surface recognize the proteins on the surface of the virus, which allows the cells to take in the virus.

Question 25

What is AVV?

Answer 25

It is a viral vector. It replicates non-chromasomally (episomally). It can be taken up into non-dividing tissues.

Question 26

What viruses can only get taken up into a dividing cell?

Answer 26

Retroviruses.

Question 27

What is a CAG promoter?

Answer 27

It is a strong promoter that has introns, exons, and an enhancer.

Question 28

How do you make a knockout mouse?

Answer 28

1. isolate stem cells from blastocyst.
2. Make plasmid that has DNA that is complementary to the gene you are knocking out, but an important part of the gene has antibiotic resistance gene.
3. Make the plasmid enter the cells via electroporation or lipid.
4. Treat cells with antibiotics that select for your cells that incorporated the plasmid.
5. Inject the cells into a blastocyst and put it in a new mouse.
6. Chimeric mice are born.
7. Mate chimeric mice with heterozygous mice.

Question 29

What is the cost-benefit balance between GEVIs and a conventional electrode?

Answer 29

A conventional electrode has better temporal resolution and GEVIs have better spatial resolution.

Question 30

What is a GEVI?

Answer 30

It is an optogenetic sensor that measures changes in voltage as a result of action potential in the soma and axon hillock, giving good spatial resolution. You can record from multiple neurons simultaneously and see which neuron are getting the signal from.

Question 31

What procedure about making a knockout is different from making a knockin?

Answer 31

In knockin mice, you don’t target a gene because you would kill its functionality. Instead you randomly insert the knockin DNA or into a part of the genome that is not needed.

Question 32

What are chimeric mice and why do you cross them with wildtype mice?

Answer 32

• Chimeric mice have some cells that came from the initial donor, and some from the mouse that gave birth. The two mother mice are different from each other.
• Mate with wildtype mice because you want to get heterozygous KO (+/-) or KI (+/0).

Question 33

When can you mate chimeric mice with wildtype mice?

Answer 33

When the chimeric mice have germline cells.

Question 34

What are the genotypes of heterozygous mice produced by chimeric and wildtype mice?

Answer 34

1/4 wildtype, 1/2 heterozygous, 1/2 homozygous.

Question 35

What is the current flow caused by that the EEG measures?

Answer 35

The current flow is caused by excitatory input which leads to calcium influx into the dendrites and soma.