Deck 13: Ways of Exploring

Question 1

What is a loss-of-function mutation?

Answer 1

The disruption of the function of a single gene without affecting any other gene in the genome.

Question 2

All of the techniques described need appropriate controls. In knockout animals, many investigators will try and 'rescue' the phenotype by adding back the knocked-out protein. Why is this a critical control and what is the expected result, in general?

Answer 2

Knockouts are generated to remove specific proteins. Even under highly controlled conditional knockouts the removal of the protein could have unintended side-effects, for example interfering with another protein. Therefore adding the protein of interest back should fully rescue the phenotype (whatever that is). If adding the protein does not rescue the phenotype then the knockout was not successful or it was not selective.

Question 3

What is one of the potential advantages that human genome sequencing will have on neuroscience?
(a) It will allow scientists to better correlate genetic phenotypes with mental illnesses.
(b) It will allow scientists to manipulate genes and cure mental illnesses.
(c) It will allow scientists to express light-activated channels into specific neurons to help cure diseases.
(d) It will allow scientists to design babies free of disease.
Genetic and Molecular Techniques

Answer 3

(A)
With the relative ease of genetic screening, correlations between diseases like mental illness and genotype will be more available. Although manipulating genes to cure diseases may be possible in the distant future it is less probable at the current time. Expressing light-activated channels requires either a viral infection of specific cells or transgenic expression. Viral infection of these channels is possible, but the addition of the appropriate light stimulation provides further problems to this invasive procedure.

Question 4

Which is/are the correct statement(s) about confocal and/or light-sheet microscopy?
(a) Confocal microscopy detects fluorescence from a single focal plane of tissue.
(b) Confocal microscopy focuses light on a specific focal plane in a section of tissue.
(c) Light-sheet microscopy detects fluorescence from a single focal plane of tissue.
(d) Light-sheet microscopy uses a large objective lens to focus light on a large sheet of tissue.
(e) Confocal microscopy is faster than light-sheet microscopy.
(f) Both require fluorescent markers in the tissue.

Question 5

One way to knock-down protein expression is by RNA-interference (RNAi).
A. What is the advantage of this system over a more common knock-out?
B. Why do you need double-stranded RNA for gene knockdown by RNAi?

Question 6

What main advantage do both Drosophila and C. elegans provide as an animal model in neuroscience?
(a) They have small neurons from which it is easy to record activity during a behavior.
(b) They are good for precise genetic manipulations that link the functions of specific genes or neuronal populations to the development and function of the nervous system.
(c) They have large, identified neurons that are easy to record from electrophysiologically.
(d) The connections of all the neurons have been mapped.

Question 7

You find a new presynaptic protein that you call 'synprotelm' and you want to see where it is in relation to the presynaptic Ca²⁺ channels. What is the best method to use to understand this? Defend your answer.
(a) Scanning electron microscopy
(b) Transmission electron microscopy
(c) Super-resolution fluorescence microscopy
(d) Immunoelectron microscopy

Question 8

Surprisingly, many people believe that animals other than vertebrates (and even mammals) are not fundamentally important to scientific research, as they are too simplistic or not similar enough to humans. Give one example of an invertebrate, other than Drosophila and C. elegans, that has demonstrated a fundamental concept in neuroscience. Include one of the advantages this animal provided.

Question 9

You are studying a new species of bird and want look at areas of the brain that are sexually dimorphic. Which technique is best for this?
(a) CLARITY
(b) Nissl stain
(c) Confocal microscopy
(d) Intracellular dye fills
(e) Electron microscopy

Question 10

To find gene(s) involved in the Drosophila Period phenotype investigators used 'forward genetics'. What does this mean?
(a) The investigators knew the Period gene was involved in circadian rhythms and so used homologous recombination to knock-out the gene and found the circadian rhythm was altered.
(b) The investigators thought the Period gene was involved in circadian rhythms and made a conditional knockout of the Period gene using the Cre/lox system. When the Period protein was knocked out the flies displayed a disrupted circadian rhythm.
(c) The investigators used random mutagenesis to find animals with the Period phenotype and used molecular genetic methods (such as positional cloning) to identify the gene.
(d) The investigators used the CRISPR/Cas9 system to selectively manipulate the Period gene and confirmed this by showing a disrupted circadian rhythm in the flies.

Question 11

How can single, identifiable neurons be labeled? Choose all that apply.
(a) Intracellular dye fill
(b) Using a thy1-promotor to drive GFP expression
(c) Anterograde tracers
(d) Retrograde traces
(e) Golgi stain
(f) Nissl stain

Question 12

What does a Nissl stain tell you? Choose all that apply.
(a) The location of neurons and glia
(b) The location of fiber tracts (axons)
(c) The connectivity between two neurons
(d) The identity of a neuron's neurotransmitter
(e) The location of cell nuclei

Question 13

The CASPR/Cas9 system is a new, very powerful technique that can knockout genes in mice, flies, and animals that are not accessible to genetic manipulation. If you are trying to remove expression of a presynaptic protein you need to add a guide RNA and Cas9. Why do you need a guide RNA? Choose all that apply.
(a) The guide RNA guides the Cas9 to the loxP site on the target DNA.
(b) The guide RNA targets the selected DNA and brings Cas9 to the DNA target site.
(c) The guide RNA guides CRISPR to the Cas9 protein to target the selected piece of DNA.
(d) CRISPR is inserted into the genome where the guide RNA guides Cas9 to the deletion site.

Question 14

You want to determine which genes are up- and down-regulated in glia after brain injury. Which technique would be best for this type of analysis?
(a) RNAseq
(b) Immunostaining for a glial specific marker
(c) Southern blot
(d) In situ hybridization
Anatomical Techniques

Question 15

An important principle in neuroethology is to select a model system with a robust behavior. Why is this an important concept when considering which animal to study?

Question 16

The CASPR/Cas9 system is a new, very powerful technique that can knockout genes in mice, flies, and animals that are not accessible to genetic manipulation. If you are trying to remove expression of a presynaptic protein you need to add a guide RNA and Cas9. What does the Cas9 do?
(a) Cas9 targets and removes the gene of interest by annealing the flanking regions.
(b) Cas9 binds to the gene of interest so that it is targeted for degradation by CASPR.
(c) Cas9 creates a double-stranded break in the DNA.
(d) Cas9 causes homologous recombination of sites around the targeted gene.

Question 17

An experiment reversed Rett symptoms by restoring MeCP2 expression in young mice. Overexpression of MeCP2 causes significant neurological defects so the investigators used an interesting technique where they inserted a transcription stop site flanked by two loxP sites in between the transcription start site and the coding region.
A. What is the advantage of this technique?
B. The investigators then used tamoxifen-inducible CreER excision of the transcription stop site. What was the function of this procedure?

Question 18

Using any vertebrate animal in research is governed by ethical rules that are regulated by government agencies and maintained through an institutional animal care and use committee. What are some of the ethical obligations of researches when using vertebrates in research?

Question 19

What is the difference between a knock-in and a knockout?

Question 20

Select all that are correct about diffusion tensor imaging (DTI).
(a) Can resolve axon trajectories throughout the whole brain
(b) Can resolve axon trajectories in major axon bundles
(c) Can resolve dendritic arborizations in the cortex
(d) Makes an estimate of water diffusion
(e) Makes an estimate of neural activity
(f) Makes an estimate of blood flow

Question 21

Which of the following are ways to activate neurons or groups of neurons? Choose all that apply.
(a) Lesions
(b) Stimulation
(c) Overexpression of Kir
(d) Expression of ChR2
(e) Expression of halorhodopsin
(f) ATP activation

Question 22

What are the advantages and disadvantages of studying an animal in its natural environment?

Question 23

What is the advantage of an extracellular recording over an intracellular recording?

Question 24

Why is functional magnetic resonance imaging advantageous for human brains? Select all that apply.
(a) It is very fast.
(b) It is noninvasive.
(c) It shows connections between brain areas.
(d) It has high spatial resolution.

Question 25

Carl von Frisch studied honeybee foraging to understand the cues bees use to transmit information about the location of a food source. What was correlated with distance of feeding place?
(a) The angle to the sun in relation to the outgoing flight
(b) The number of times the bee turned to the left or right
(c) The total duration of the tail wag
(d) The number of bees that went to a particular location

Question 26

In the experiment in Figure Q13-38, the motor neuron of a muscle expressed the P2X₂ transgene and the response in the muscle was recorded. Why did application of ATP activate the muscle? Figure Q13-38
(a) Addition of ATP activated of the P2X₂ channel which directly increased transmitter release by moving vesicles to the presynaptic membrane.
(b) Addition of ATP resulted in opening of the P2X₂ channel and an influx of sodium, which activated the motor neuron, releasing neurotransmitter and activation of the muscle.
(c) Addition of the resulted in activation of the P2X₂ protein, a G protein coupled receptor. Once activated the receptor increased the probability of neurotransmitter release and activated the muscle.
(d) Addition of ATP resulted in activation of the ATP receptor, a G protein coupled receptor. Once activated, this G protein indirectly activated P2X₂, which increased the probability of neurotransmitter release from the motor neuron and activated the muscle.

Question 27

Deep brain stimulation is a technique used to help people with different diseases, including Parkinson's. People with Parkinson's disease have difficulties with movement due to a decrease in dopaminergic input to the basal ganglia. For Parkinson's patients the subthalamic nucleus (STN) is stimulated and this relieves some of the symptoms associated with the disease. However, it is not clear if stimulation of the nucleus stimulates neurons within the STN, or inputs to the STN (from the cortex). Outline an experiment in a mouse that could look at the difference between activation of the STN and activation of inputs to the STN.
Behavioral Analyses

Question 28

How does halorhodopsin (NpHR) inactivate neurons?
(a) Halorhodopsin is activated by blue light, which causes a nonselective cation current which allows more K⁺ efflux than Na⁺ influx, hyperpolarizing the neuron.
(b) Halorhodopsin is activated by blue light, which results in increased permeability to chloride, effectively silencing the cells.
(c) Halorhodopsin is activated by yellow light, which causes a nonselective cation current which allows more K⁺ efflux than Na⁺ influx, hyperpolarizing the neuron.
(d) Halorhodopsin is activated by yellow light, which results in an increased transportation of chloride across the membrane, effectively silencing the cells.

Question 29

What is a connectome?
(a) A wiring diagram of synaptically connected neurons
(b) A map of all the neurons in a specific nucleus
(c) All the connections that a single neuron makes
(d) The complex interactions in a molecular pathway
(e) Results from diffusion tensor imaging

Question 30

How does ChR2 activate neurons?
(a) Blue light causes a conformational change in a rhodopsin protein which causes the channel to open. This results in a nonselective cation current that allows more Na⁺ influx than K⁺ efflux, depolarizing the neuron.
(b) Blue light causes a conformational change in a rhodopsin protein which causes the channel to open. This results in a nonselective cation current that allows more K⁺ efflux than Na⁺ influx, depolarizing the neuron.
(c) Blue light activates the rhodopsin protein that activates a G protein which opens a nonselective cation current which allows more Na⁺ influx than K⁺ efflux, depolarizing the neuron.
(d) Blue light activates the rhodopsin protein that activates a G protein which opens a nonselective cation current which allows more Na⁺ influx than K⁺ efflux, hyperpolarizing the neuron.
(e) Blue light activates the rhodopsin protein that activates a G protein which opens a nonselective cation current which allows more K⁺ efflux than Na⁺ influx, depolarizing the neuron.
(f) Blue light activates the rhodopsin protein that activates a G protein which opens a nonselective cation current which allows more K+ efflux than Na+ influx, hyperpolarizing the neuron.

Question 31

Neurons in the motor cortex are broadly tuned and show selectivity for a direction of movement. There is evidence that movement is coded by the activity of a population of neurons. One of the experiments that provided evidence for this used a multi-electrode array. Why is this a good method for studying this type of coding and why would a local field potential or intracellular recording not have provided the evidence needed to show a population code?

Question 32

What does functional magnetic resonance imaging measure?
(a) The synchronous electrical activity of thousands of neurons
(b) The blood flow near excited neurons
(c) The activity of glia near excited neurons
(d) The magnetic activity of electrically excited neurons

Question 33

What is the advantage of an intracellular recording over an extracellular recording?

Question 34

Two neurons, A and B, are reciprocally connected. To determine how they are connected to each other you stimulate one while recording from the other with the results show in Figure Q13-35. When you stimulate A, you see the response on the left. When you stimulate B, you see the response in the middle. In addition, when you hyperpolarize A, you see a small hyperpolarization in B. Based on these results, which circuit is correct? Figure Q13-35

Question 35

Which of the following are ways to inactivate neurons or group of neurons? Choose all that apply.
(a) Lesions
(b) Stimulation
(c) Overexpression of Kir
(d) Expression of ChR2
(e) Expression of halorhodopsin
(f) ATP activation

Question 36

Trans-synaptic tracing uses rabies virus to spread across synapses. In the scenario in Figure Q13-24, what would happen if the presynaptic cell also expressed the glycoprotein transgene? Figure Q13-24
(a) The virus spread would not be limited to presynaptic neurons of the starter cells.
(b) The virus would not be limited to how many neurons it could infect.
(c) The virus would not be able to infect other synaptic partners and would stay in the neuron presynaptic to the starter neuron.
(d) The virus would not be able to spread to any synaptic partners and would stay in the starter cell.

Question 37

You are recording from some neurons in the pulvinar nucleus in the thalamus and want to know where neurons in that nucleus project. What is/are the best method(s) to use to determine this? Select all that apply.
(a) Use a retrograde tracer in the pulvinar nucleus
(b) Use a retrograde tracer in the cortex
(c) Use an anterograde tracer in the pulvinar nucleus
(d) Use an anterograde trace in the cortex
(e) Use diffusion tensor imaging (DTI)

Question 38

True/False. Making a complete map of all the connections in a circuit is sufficient to tell you how the circuit works. Defend your answer.
Recording and Manipulating Neuronal Activity

Question 39

The chapter started with a quote from Sydney Brenner "Progress in science depends on techniques, new discoveries, and new ideas, probably in that order." Based on what you have read in this chapter and the entire textbook do you agree or disagree with this statement??

Question 40

You are a new investigator at a lab and think you have identified an area of the brain (fictitiously called SASD) important for social interactions, which might be effected in a mouse model of autism spectrum disorder. Describe an experiment in which you test the necessity and sufficiency of this brain area in social interactions. Include the neural manipulation and behavioral assay. Also include the predicted outcome of the experiment if you are correct.

Question 41

Why is a closed-loop design so important for understanding the neural basis of behavior?