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Neuroscience Y2S1 > Neuroscience Research Methods 2 > Flashcards

Neuroscience Research Methods 2 Flashcards

1
Q

What is Western Blotting used for in neuroscience research? (1)

A

Separating and identifying proteins in a tissue sample or serum.

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2
Q

Briefly describe the principles of performing Western Blotting. (5)

A
  • Homogenise tissue sample
  • Denature proteins (heat with chemicals)
  • Separate proteins using an acrylamide gel and electrophoresis
  • Transfer proteins to nylon membrane
  • Detect proteins with antibodies
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3
Q

Name the essential steps used to perform immunohistochemistry. (7)

*There are eleven total steps, but the rest are only performed under specific circumstances

A
  • Tissue preparation
  • Sectioning and mounting on glass slides
  • Membrane permeabilization
  • Blocking
  • Primary antibody incubation
  • Secondary antibody incubation
  • Detection and counterstaining
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4
Q

What is immunohistochemistry used for in neuroscience research? (1)

A

Identify/localise specific tissue components using specific antigen/antibody binding.

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5
Q

Describe the difference between immunohistochemistry and immunocytochemistry. (1)

A

IHC performed on tissue samples,

ICC performed on cells (eg. in culture).

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6
Q

Describe what positive control would be used for immunohistochemistry. (1)

A

A tissue that is known to express the antigen.

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7
Q

Describe what negative control would be used for immunohistochemistry. (1)

What things would the negative control pick up in immunohistochemistry? (2)

A

Same steps but with no primary antibody.

Picks up non-specific binding and endogenous peroxidases.

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8
Q

Give two general ways that tissue integrity can be preserved before immunohistochemistry is performed. (2)

A

Freezing

Fixing

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9
Q

Give two ways that tissues can be frozen to preserve integrity before immunohistochemistry is performed. (2)

What temperature are tissues frozen at? (1)

A
  • Dry ice
  • Liquid nitrogen

Tissues frozen at -80C

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10
Q

Give one advantage and one disadvantage of preserving tissue integrity by freezing before performing immunohistochemistry. (2)

A

ADVANTAGE:
- Antigens remain unaltered

DISADVANTAGE:
- Defrosting before staining might destroy tissue integrity

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11
Q

Give three ways of fixing samples to preserve tissue integrity before performing immunohistochemistry. (3)

A
  • Perfusion
  • Post-fixation
  • Paraffin embedding
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12
Q

Describe the process of perfusion to fix a tissue and preserve integrity before performing immunohistochemistry. (1)

A

Inject tissue to wash out blood, and then perfuse with paraformaldehyde.

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13
Q

Describe the process of post-fixation to fix a tissue and preserve integrity before performing immunohistochemistry. (1)

What temperature do samples have to be kept at? (1)

A

Take biopsy or deceased organism, then leave in fixative overnight.

Keep samples at 4C.

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14
Q

Name two chemicals that can be used for post-fixation to preserve tissue integrity before performing immunohistochemistry. (2)

A

Methanol

Formalin

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15
Q

Describe the process of paraffin embedding to fix a tissue and preserve integrity before performing immunohistochemistry. (2)

A

Dehydrate tissue with ethanol

then immerse in paraffin wax.

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16
Q

Give two advantages and one disadvantage of preserving tissue integrity by fixing before performing immunohistochemistry. (3)

A

ADVANTAGES:
- More stable
- Provides good architecture

DISADVANTAGES:
- Might mask antigens, so may need antigen retrieval

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17
Q

True or false? (1)

If a tissue sample has been fixed to preserve integrity before immunohistochemistry, samples are best stored at -20C.

A

False - tissues CAN be stored at -20C, but only if cryoprotected

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18
Q

True or false? (1)

Sectioning and mounting must be performed on samples before performing both immunohistochemistry and immunocytochemistry.

A

False - only needs to be done when using tissue (IHC)

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19
Q

Give two methods of slicing/sectioning frozen tissue samples before performing immunohistochemistry. (2)

A
  • Cryostat
  • Freezing microtome
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20
Q

Give four methods of slicing/sectioning fixed tissue samples before performing immunohistochemistry. (4)

State any conditions required for using particular methods of sectioning.

A
  • Vibratome
  • Microtome
  • Freezing microtome (only if tissue has been cryoprotected)
  • Cryostat (only if tissue has been cryoprotected)
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21
Q

Which out of vibratome/microtome is able to produce thinner slices when sectioning tissue samples? (1)

A

Microtome

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22
Q

True or false? (1)

A disadvantage of preserving tissue integrity by freezing before performing immunohistochemistry is that when sectioning the tissue, you will be unable to work at room temperature.

A

True

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23
Q

When preparing to perform immunohistochemistry on a tissue sample, under what circumstances would deparaffinisation and rehydration need to be carried out? (1)

When would this step be carried out? (1)

A

If the sample has been paraffin embedded to preserve tissue integrity.

Done after tissue has been cut with microtome but before antigen retrieval/membrane permeabilization.

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24
Q

Describe how you would deparaffinise and rehydrate a tissue sample which has been paraffin embedded, before performing immunohistochemistry. (2)

A
  • Remove embedded was by immersing in organic solvents (xylene)
  • Rehydrate by immersing in alcohol solutions with increasing percentage of H2O
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25
Q

When performing immunohistochemistry, in what circumstances would antigen retrieval need to be performed? (1)

A

If the tissue integrity has been preserved by fixing (as opposed to freezing)

(as the fixative bonds mask antigens)

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26
Q

Give three possible methods of antigen retrieval, which may have to be performed before immunohistochemistry. (3)

A
  • High temperature
  • Enzymes
  • HCl
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27
Q

Describe how antigen retrieval can be performed using high temperatures in immunohistochemistry. (2)

Give a disadvantage of this technique. (1)

A
  • Incubate samples with EDTA/citrate buffer
  • at high temperature (80-99C)

However must be calibrated for each antigen as may need different concentrations of buffer and/or temperature.

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28
Q

Name two potential enzymes that could be used for enzymatic antigen retrieval in immunohistochemistry. (2)

What is a disadvantage of enzymatic antigen retrieval? (1)

A
  • Trypsin
  • Protease

DISADVANTAGE:
Bad for morphology and may destroy some epitopes.

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29
Q

Describe the HCl method of antigen retrieval in immunohistochemistry. (1)

A

Incubate sections with 1N and then 2N HCl

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30
Q

Why is membrane permeabilization required when performing immunohistochemistry? (1)

A

To be able to stain intracellular or membrane-spanning antigens.

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31
Q

Give three possible methods of membrane permeabilization when performing immunohistochemistry. (3)

Which is the most popular?

A
  • Detergents (most popular)
  • Acetone/methanol
  • Liquid nitrogen
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32
Q

Give two possible detergents that can be used for membrane permeabilization when performing immunohistochemistry. (2)

In what circumstances can detergents not be used for membrane permeabilization? (1)

A
  • Triton-X
  • Tween

Cannot be used if you want to detect membrane proteins.

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33
Q

Under what circumstances is liquid nitrogen used for membrane permeabilization when performing immunohistochemistry? (1)

How does liquid nitrogen permeabilise membranes? (2)

A

Used when electron microscopy is going to be performed.

Quick freezing of tissue forms tiny holes in the cell membrane.

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34
Q

How is acetone/methanol able to permeabilize membranes when performing immunohistochemistry? (1)

A

Able to precipitate proteins outside cell membranes.

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35
Q

In what circumstances would endogenous peroxidase inactivation have to be performed when doing immunohistochemistry? (1)

A

If using the horseradish peroxidase enzyme to visualise antigens - as tissues contain endogenous peroxidases which would give non-specific background noise

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36
Q

How is endogenous peroxidase inactivation carried out when performing immunohistochemistry? (1)

A

Usually done using 0.5-3% H2O2 dissolved in PBS or methanol.

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37
Q

Why does blocking have to be performed during immunohistochemistry? (3)

A

To remove any non-specific binding eg. binding between charged molecules

which would produce background noise.

The blocking agent will bind and block potential non-specific binding sites.

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38
Q

Give three potential blocking agents that could be used to reduce non-specific binding during immunohistochemistry. (3)

A
  • Normal serum from secondary antibody species (obtained before antigen was introduced)
  • BSA (bovine serum albumin)
  • Fish gelatin
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39
Q

Describe one advantage and one disadvantage of carrying out primary antibody incubation at 37C for 1h as opposed to 4C for 3 days when performing immunohistochemistry. (2)

Also give an advantage of performing primary incubation for a longer time but at a lower temperature. (1)

A

ADVANTAGE:
- Higher signal

DISADVANTAGE:
- Higher background

Incubating for longer at lower temperature improves sensitivity without increasing background noise.

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40
Q

During which antibody incubation of immunohistochemistry is it usually essential to calibrate for the specific antibody being used? (1)

Why is this needed? (1)

A

Primary incubation

Needed because you might need different concentrations of antibody or different conditions, depending on the antibody being added.

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41
Q

True or false? (1)

In immunohistochemistry, when performing the secondary antibody incubation, it is essential to incubate for longer than with the primary antibody.

A

False - with secondary antibody, incubation is usually shorter than that for primary antibody

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42
Q

Why is a secondary antibody used in immunohistochemistry as well as a primary antibody? (1)

A

Just using a primary antibody produces more background noise, as there is more chance of non-specific binding.

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43
Q

In immunohistochemistry, primary antibodies are raised against the epitope of interest.

What are secondary antibodies raised against? (1)

Use an example to help explain.

A

The species that the first antibody was obtained from.

eg. if mouse antibodies used as primary antibody, secondary antibody is anti-mouse, but would have to be obtained from a different species such as goat

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44
Q

Why may signal boosting be performed during immunohistochemistry? (1)

A

It is a way of increasing signal/noise ratio and sensitivity.

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45
Q

Describe the biotin method of signal boosting in immunohistochemistry. (4)

A
  • Secondary antibody is biotinilated (biotin groups added to Ab)
  • Avidin or streptavidin molecules (able to bind to many biotin molecules) mixed with secondary antibody
  • More biotin (with added enzyme) added to secondary Ab mixture to form complexes of avidin-biotin-enzyme attached to antibodies
  • Abs added to immunohistochemistry sample and signal amplified because many enzymes bound to one secondary antibody
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46
Q

Describe the anti-peroxidase method of signal boosting in immunohistochemistry. (4)

A
  • Secondary antibody is peroxidase
  • Anti-peroxidase enzymes bind to secondary antibody
  • Which then binds more peroxidase
  • Which results in many enzyme molecules per secondary antibody so signal is boosted
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47
Q

Give three general methods of antibody detection when performing immunohistochemistry. (3)

A
  • Fluorochromes
  • Enzymes
  • Electron-scattering compounds
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48
Q

In what circumstances would electron-scattering compounds be used to detect antibodies in immunohistochemistry? (1)

Suggest a compound which could be used. (1)

A

When electron microscopy is being performed.

Ferritin colloidal gold

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49
Q

Give two examples of enzymes which could be conjugated to secondary antibodies for detection in immunohistochemistry. (2)

How do these enzymes allow detection of molecules? (1)

A
  • Peroxidase (horseradish)
  • Alkaline phosphatase

The enzymes change the colour of a substrate - more secondary enzyme = more colour change

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50
Q

Give two advantages and one disadvantage of using enzymes to detect antibody binding in immunohistochemistry. (3)

A

ADVANTAGES:
- Brightfield microscopes sufficient for analysis
- Once stained, the specimens have an unlimited shelf life

DISADVANTAGES:
- Substrate reagents are often toxic/carcinogenic

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51
Q

Give two examples of fluorochromes which could be conjugated to secondary antibodies for detection in immunohistochemistry. (2)

A
  • Fluorescein
  • Rhodamine
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52
Q

Give two advantages and two disadvantages of using fluorochromes to detect antibody binding in immunohistochemistry. (4)

A

ADVANTAGES:
- Enables double and triple labelling
- Higher resolution

DISADVANTAGES:
- Requires special fluorescent microscopes
- Signal decays with time (have to store slides in dark until viewing)

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53
Q

Why is counterstaining performed during immunohistochemistry? (1)

A

Allows other structures in the tissue to be identified, so the location of specific signals can be determined.

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54
Q

Give two counterstains (and the structures that they identify) which can be used for colourimetric (enzyme) reactions in immunohistochemistry. (2)

A

Nissl (stains DNA/RNA)

Haematoxylin-eosin (stains cytoplasm & nucleus)

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55
Q

Give two counterstains (and the structures that they identify) which can be used for fluorescent reactions in immunohistochemistry. (2)

A

DAPI (stains A-T regions of DNA)

Hoechst (stains DNA)

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56
Q

What is meant by epigenetics? (2)

A

Heritable, but potentially reversible, changes in gene expression

that occur without changes in the DNA sequence.

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57
Q

What is the main effect of DNA methylation on gene transcription? (1)

A

Inhibits gene transcription

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58
Q

In epigenetics, where exactly on the DNA molecule are methyl groups added? (1)

A

The cytosine part of CpG dinucleotides.

*CpG dinucleotide = cytosine adjacent to guanine, connected by phosphodiester bond

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59
Q

Describe the molecular mechanism by which DNA methylation inhibits DNA transcription. (2)

A
  • Proteins that bind to methylated DNA (readers of DNA methylation) exclude transcriptional machinery from accessing DNA promotor region
  • DNA methylation readers can also recruit large protein complexes, including histone deacetylases, which further shut down gene expression
60
Q

Name two molecules which are readers of DNA methylation. (2)

  • Also called methylcytosine-binding proteins
A
  • MeCP2
  • MBD1-4
61
Q

Describe what is meant by a CpG island, in the context of DNA methylation. (2)

A

Areas of enriched CpG dinucleotides

which occur at DNA promotor regions.

62
Q

CpG dinucleotides occur lots in DNA promotor regions, but they also occur in intergenic regions.

In intergenic regions, are the CpG dinucleotides more likely to be methylated or unmethylated? (1)

Same question regarding promotor regions. (1)

A

Intergenic regions = methylated

Promotor regions = unmethylated

63
Q

Name two DNA methyltransferases which catalyse DNA methylation. (2)

In what circumstances do each of these enzymes catalyse DNA methylation? (2)

A

DNMT1 (copies methylation patterns during mitosis)

DNMT3a/3b (‘de novo’ methyltransferases which add new methyl groups to DNA)

64
Q

Name five epigenetic ways in which histones can be modified. (5)

A
  • Acetylation
  • Methylation
  • Phosphorylation
  • Ubiquitinylation
  • SUMOylation
65
Q

What is a histone? (1)

A

A protein which DNA wraps around. It provides structural support for a chromosome.

66
Q

Describe the structure of an individual histone. (1)

A

Globular protein plus a tail.

67
Q

Describe the structure of a nucleosome. (2)

A

8 histones + DNA

Histones named H2A, H2B, H3, H4

68
Q

Name the two histones contained within a nucleosome which are the most important in terms of epigenetics. (2)

A

H3
H4

69
Q

Describe how histone acetylation alters gene expression. (3)

A

Activates gene transcription

by binding to lysine residues on histone tail.

This alters the charge on lysine and therefore its binding properties.

70
Q

Name the enzyme which catalyses histone acetylation. (1)

A

Histone acetyltransferases (HAT)

71
Q

Name the enzyme which catalyses histone deacetylation. (1)

A

Histone deacetylases (HDAC)

72
Q

Describe how histone methylation alters gene transcription. (2)

A
  • Methyl groups added to arginine and lysine residues on histone tail
  • Can activate or deactivate genes depending on the specific residue it binds to
73
Q

True or false? (1)

Histone acetylation and methylation are the more common epigenetic histone modifications.
Out of the two, acetylation is the most stable.

A

False - methylation is the most stable

74
Q

In epigenetics, on which histone residues does phosphorylation occur? (2)

What enzyme catalyses this? (1)

A

Serine and threonine residues

Catalysed by kinases

75
Q

Does histone phosphorylation activate or inhibit chromatin? (1)

A

Associated with both

76
Q

In epigenetics, large ubiquitin and SUMO residues can be added to which amino acids on the histone tail to cause changes in gene expression? (1)

A

Lysine

77
Q

Describe the difference between euchromatin and heterochromatin, making reference to gene transcription. (2)

A

HETEROCHROMATIN:
- Closed
- Inaccessible to transcriptional machinery

EUCHROMATIN:
- Open
- Genes can be transcribed

78
Q

How can non-coding strands of RNA affect gene expression? (1)

Give three examples of non-coding RNA which can do this. (3)

A

Silence genes

  • MicroRNAs
  • Piwi-interacting RNAs
  • Long non-coding RNAs
79
Q

True or false? (1)

MicroRNAs are endogenous, double-stranded RNA molecules which result in pre-transcriptional gene silencing. They are involved in development, differentiation, and disease.

A

False - they are single-stranded, and they result in post-transcriptional gene silencing

80
Q

Describe how piwi-interacting RNAs are able to cause gene silencing. (2)

A

Control transposable elements (bits of DNA that move from one area to another)

and are able to direct DNA methylation at transposable elements.

81
Q

Describe briefly how long non-coding RNAs may play a role in gene silencing. (1)

A

May direct epigenetic enzymes to sites in the genome.

82
Q

What is bisulfite sequencing used for in neuroscience research? (1)

A

Measuring DNA methylation

83
Q

Describe how bisulfite sequencing can be used to measure DNA methylation. (4)

A
  • Bisulfite converts UNMETHYLATED cytosines to uracil
  • Methylated cytosines remain unchanged
  • PCR amplifies DNA and converts uracils to thymine
  • Sequencing can then be used to compare the new sequence with the original sequence (or a reference genome)
84
Q

What is the MeDIP technique used for in neuroscience research? (1)

A

Measuring DNA methylation

85
Q

Briefly describe how MeDIP is performed. (4)

A
  • Isolate DNA from sample
  • Sonicate DNA with high frequency to break into fragments
  • Precipitate methylated DNA fragments using immunoprecipitation (use antibodies which bind to methyl groups)
  • Digest antibodies and use qPCR to quantify results for gene of interest
86
Q

Describe how you would interpret MeDIP/ChIP results. (4)

A

Calculate Ct for precipitated (methylated) DNA sample

Calculate Ct for original DNA before the methylated parts were isolated (this is known as the input)

If Ct same for both samples, 100% gene copies are methylated

If Ct of MeDIP/ChIP is higher than input, only a few copies of the gene are methylated
(Ct input 19 & Ct MeDIP/ChIP 20 = 50% gene copies are methylated)

87
Q

What is the ChIP technique used for in neuroscience research? (1)

A

Measuring histone modifications

88
Q

Briefly describe how ChIP is performed. (3)

A
  • Sonicate tissue with high frequency to break into fragments (DNA still wound around histones)
  • Precipitate methylated histones and DNA fragments using immunoprecipitation (use antibodies which bind to methyl groups)
  • Dissociate DNA from histones and use qPCR to quantify results for gene of interest
89
Q

Optical imaging uses which types of electromagnetic waves? (3)

Name two categories of optical imaging. (2)

A
  • Infrared
  • Visible light
  • Ultraviolet

Light microscopy and fluorescent microscopy

90
Q

What types of structures might be visualised using electron microscopy? (1)

Give a limitation of this technique. (1)

A

Small structures - subcellular and molecular (<10um)

Limitation - samples require processing so cannot be used in vivo

91
Q

Give three general applications of fluorescent microscopy in neuroscience research. (3)

A
  • Localisation
  • Dynamics (changes over time)
  • Signalling
92
Q

Give three examples of what is meant by the following sentence:

‘In neuroscience research, fluorescent microscopy can be used to show dynamic changes in a tissue.’

(3)

A
  • Can show structural changes, for example cell morphology or synaptic plasticity
  • Can show expressive changes, for example up/down regulation of receptors
  • Can show transport, for example internalisation and intracellular trafficking
93
Q

Describe how fluorescence works. (3)

A
  • Fluorescent molecules absorb photons of a particular wavelength
  • Use energy to excite electrons to higher energy state
  • Then release light of a specific wavelength as the electrons return to ground state
94
Q

In fluorescent microscopy, describe what is meant by ‘excitation wavelength’ and ‘emission wavelength’. (2)

A

EXCITATION WAVELENGTH is the light that the fluorophore absorbs.

EMISSION WAVELENGTH is the light that the fluorophore gives off.

95
Q

True or false? (1)

In fluorescent microscopy, the excitation wavelength is always longer than the emission wavelength, and the two wavelengths must overlap to allow adequate detection.

A

False:

  • excitation wavelength SHORTER (higher energy) than emission wavelength
  • Ideally, excitation and emission wavelengths SHOULD NOT OVERLAP
96
Q

What colour on the visible light spectrum has the longest wavelength and which has the shortest? (2)

Which one has higher energy? (1)

A

Longest - red

Shortest - purple

Shorter wavelength (purple) has higher energy

97
Q

When performing co-localisation in fluorescent microscopy, why is it important that the emission wavelengths of the fluorophores chosen have as little overlap as possible? (1)

A

So that two distinct colours can be visualised

98
Q

In fluorescent microscopy, describe what co-localisation is used for. (1)

A

Used to determine if 2 molecules are expressed together or in similar places.

99
Q

Describe the results seen in co-localisation if two molecules are expressed in the same place. (2)

A

An emission wavelength ‘between’ the 2 individual ones (eg. yellow, if the individual ones were red and green) are seen if the molecules are expressed together.

This is seen by the merge images.

100
Q

Name two types of fluorescent microscope. (2)

A

Widefield

Confocal

101
Q

Give one advantage and one disadvantage of using widefield microscopy as opposed to confocal microscopy. (2)

A

ADVANTAGE:
- More simple

DISADVANTAGE:
- Lower-quality image produced

102
Q

Describe what confocal microscopy is used for. (2)

A

Produces a clear image with less background noise

of thin sections of a thick sample.

ie. can penetrate to different depths of the sample to create 2D or 3D images

103
Q

Name the ‘steps’ or ‘parts’ of the fluorescent widefield microscope in order. (9)

A
  • Light source
  • Excitation filter
  • Dichroic mirror
  • Objective lens
  • Specimen
  • Dichroic mirror
  • Emission filter
  • Ocular lens
  • Detector
104
Q

Name the ‘steps’ or ‘parts’ of the fluorescent confocal microscope in order. (10)

A
  • Laser light source
  • Pinhole aperture
  • Excitation filter
  • Dichroic mirror
  • Objective lens
  • Specimen
  • Dichroic mirror
  • Emission filter
  • Detector pinhole aperture
  • PTM detector
105
Q

Describe what a dichroic mirror is for and how it works in fluorescent microscopy. (2)

A

Reflects light waves below a specific wavelength and lets all other wavelengths pass through.

So it directs excitation waves to sample and lets emission waves through to detector.

106
Q

Describe the different light sources used in fluorescent widefield and confocal microscopy. (2)

A

WIDEFIELD:
- uses a normal focussed source like a bulb

CONFOCAL:
- uses laser to produce high-energy, narrow beam of light

107
Q

Why are pinhole apertures used during fluorescent confocal microscopy? (1)

A

To focus light and produce a uniform illumination beam.

108
Q

What is the role of the excitation filter in fluorescent widefield and confocal microscopy? (1)

A

Restricts light to a certain wavelength range (lets excitation wavelength through)

109
Q

What is the role of the objective lens in fluorescent widefield and confocal microscopy? (1)

A

Focus light onto the sample

110
Q

What is the role of the emission filter in fluorescent widefield and confocal microscopy? (1)

A

Lets emitted wavelengths through only so removes light which hasn’t come from the fluorophore.

111
Q

What is the role of the ocular lens in fluorescent widefield and confocal microscopy? (1)

A

Magnify image

112
Q

What is the role of the PTM detector in fluorescent confocal microscopy? (1)

A

Camera which detects image produced

113
Q

Describe what can be achieved by moving the focal plane during fluorescent confocal microscopy. (2)

A

Produce a Z axis

so the light can penetrate to different layers of the sample.

114
Q

Give four general ways of fluorescently labelling sample tissue. (4)

A
  • Direct binding fluorophores
  • Antibodies
  • Bioluminescence
  • Fluorescent indicators
115
Q

Describe what is meant by ‘direct binding fluorophores’. (1)

A

Fluorescent molecules which bind directly to specific cell components, so are not generally used for specific proteins.

116
Q

Name three examples of direct binding fluorophores. (3)

A
  • DAPI
  • Phalloidin
  • Fluorescein
117
Q

What cellular component does DAPI bind to? (1)

What type of molecules is it? (ie. toxin, dye, antibody) (1)

Under what circumstances can DAPI be used? (ie. in vitro / in vivo / fixed tissue) (1)

A

Binds to DNA

Dye molecule

Can be used in vitro or fixed tissue

118
Q

What cellular component does phalloidin bind to? (1)

What type of molecules is it? (ie. toxin, dye, antibody) (1)

Under what circumstances can phalloidin be used? (ie. in vitro / in vivo / fixed tissue) (1)

A

Binds to actin

Toxin

Used in vitro or fixed tissue

119
Q

What cellular component does fluorescein bind to? (1)

What type of molecules is it? (ie. toxin, dye, antibody) (1)

Under what circumstances can fluorescein be used? (ie. in vitro / in vivo / fixed tissue) (1)

A

Accumulates in tumour tissue

Dye molecule

Used in vivo during brain surgery

120
Q

Describe how we can fluorescently label a tissue sample using antibodies. (1)

Which other neuroscience research technique is this performed in? (1)

Give a disadvantage of this fluorescent labelling method. (1)

A

Conjugate fluorophore with antibody specific for protein of interest.

Used in immunohistochemistry

Cannot be done in vivo

121
Q

Describe what is meant by bioluminescence. (1)

Give two examples. (2)

A

Naturally occurring light from specific organisms

GFP from Aequorea Victoria

luciferase from fireflies

122
Q

Describe how GFP/luciferase can be used to fluorescently label tissue samples in vivo. (2)

A

GFP/luciferase gene placed under control of a promotor for a specific protein

when/if specific protein is produced, GFP/luciferase is also produced.

123
Q

Describe what is meant by a fluorescent indicator, and what they can be used for. (2)

Can this be done in vivo / in vitro / ex vivo etc? (1)

A

Fluorescent sensors which are designed to change fluorescence when they bind to a cellular component.

Can be used to see changes in activity and cellular signalling molecules, and can also detect voltage changes.

Can be done in vivo or ex vivo/in vitro

124
Q

Give an example of when a fluorescent indicator is used in neuroscience research. (1)

A

fluorescent calcium imaging

125
Q

Name a fluorescent calcium signalling molecule commonly used in neuroscience research. (1)

A

Fluo-4

126
Q

Describe how fluo-4 can be used as a fluorescent calcium signalling indicator. (4)

A
  • Positive calcium ions bind to negative Fluo-4 molecules
  • This causes conformational change in Fluo-4
  • Results in green fluorescence of fluo-4
  • Calcium binding is reversible so can detect subsequent decreases of calcium as well as increases
127
Q

Give three applications of fluorescent calcium imaging in neuroscience research. (3)

A
  • Monitor/identify neurotransmission
  • Spatiotemporal mapping
  • Pharmacological drug screening
128
Q

Briefly describe an example of how fluorescent calcium imaging can be used to monitor/identify neurotransmission. (2)

A

Release glutamate

If calcium increases in target cell, the target cell can respond to glutamate

129
Q

Briefly describe an example of how fluorescent calcium imaging can be used for spatiotemporal mapping in neuroscience research. (1)

A

Can look at calcium signalling over space and time,

eg. glial cell calcium waves

130
Q

Briefly describe an example of how fluorescent calcium imaging can be used for pharmacological drug screening. (1)

A

Can look at response of cells to drug and produce dose-response curve to find appropriate drug concentrations/doses.

131
Q

Briefly describe what is meant by optogenetics. (1)

A

Using light to stimulate cells or mediate cellular activity.

132
Q

What is rhodopsin? (1)

Name its two components. (2)

A

GPCR

Made up of opsin and retinal

133
Q

The rhodopsin receptor is made up of opsin and retinal.

What is opsin? (1)

What is retinal? (1)

A

OPSIN:
- Microbial protein which allows transmembrane movement of ions

RETINAL:
- Light-sensitive chromophore which isomerises due to light to produce response in opsin

134
Q

Name a technique which could be used to express opsins in cells which would not normally express opsins. (1)

A

Viral transfection

135
Q

Name three different types of receptor which could be expressed in cells and then activated using optogenetic techniques. (3)

A
  • Channel rhodopsin (ChR)
  • Anion selective channel rhodopsin (ACR)
  • Light-sensitive GPCR
136
Q

In optogenetics, what is channel rhodopsin (ChR), and what happens when it is activated by light? (2)

A

Light-activated cation channel

which causes depolarisation when activated by light.

137
Q

In optogenetics, what is channel anion selective rhodopsin (ACR), and what happens when it is activated by light? (2)

A

Light-activated chloride channel

which causes hyperpolarisation when activated by light.

138
Q

True or false? (1)

In optogenetics, light-sensitive GPCRs can be naturally occurring or genetically engineered.

A

True

139
Q

What is the name given to a genetically-engineered light-sensitive GPCR used in optogenetics? (1)

A

Opto-XR

140
Q

Give two general questions in neuroscience research which may be answered using optogenetics. (2)

A
  • Are specific cells, brain regions, or pathways necessary for behaviour?
  • How can we better understand and treat disease?
141
Q

In general, how can optogenetics be used to determine the necessity or role of certain neural pathways in behaviour? (1)

A

Use either depolarising or hyperpolarising rhodopsin channels to alter activity of a pathway and see response.

142
Q

vLPO neurones are GABAergic neurones which normally reduce core body temperature and activity levels.

What would be the effect of expressing and activating channel rhodopsins (ChR2) in vLPO neurones? (1)

A
  • Reduced core body temperature and activity
143
Q

vLPO neurones are GABAergic neurones which normally reduce core body temperature and activity levels.

What would be the effect of expressing and activating anion selective channel rhodopsins (hGtACR1) in vLPO neurones? (1)

A
  • Increased core body temperature and activity
144
Q

Briefly describe how optogenetics may help to better understand and treat diseases such as epilepsy. (2)

A
  • Can express and activate ChR in neurones and observe whether seizure discharges are produced
  • Can express and activate ACR in neurones and observe whether seizure discharges are reduced
145
Q

Give an example of how optogenetics can be used to influence spatiotemporal control of receptor signalling. (4)

HINT: opto-MOR

A
  • Chimeric u opioid receptor (GPCR) produced (opto-MOR) which responds to light
  • In culture, opto-MOR activation with light reduces cAMP production, similar to native opioid binding
  • Also, expressing opto-MOR in VTA results in reward-seeking behaviour
  • Can opto-MOR be used for pain relief?

Neuroscience Y2S1 (16 decks)

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