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Developmental Neuro > Exam 1 - Lecture 1: Development Techniques > Flashcards

Exam 1 - Lecture 1: Development Techniques Flashcards

1
Q

What are common animal models of neurodevelopment?

A

mouse, zebrafish, chicken, frog, fruitfly, and nematode

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2
Q

Ways to study human brain development

A

Human cell cultures, cerebral organoids, nonivasive imaging, xenografts

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3
Q

Postmortem Tissue Benefits and Weaknesses

A

Benefits: well preserved anatomy, contains informative molecules, proteins, high resolution
Weaknesses: dead, unique life experiences

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4
Q

Human Cell Culture Definition

A

Dissasociated immortalized cells growing in a dish

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5
Q

Human Cell Culture Strengths and Weaknesses

A

Strengths: able to study live human neurons and many types of experimental manipulation
Weaknesses: about as far from real 3D tissue envrionment as it gets

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6
Q

Cerebral Organoids Definition

A

3D tissue culture from human neural stem cells

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7
Q

Cerebral Organoids Strengths and Weaknesses

A

Strengths: able to study live human neurons and many types of experimental manipulation
Weaknesses: may not recapitulate some aspects of normal development

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8
Q

Non-invasive imaging (MRI, PET scans) Strengths and weaknesses

A

Strengths: able to study live human brain over time in the same individual, some experimental manipulation
Weaknesses: low resolution

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9
Q

Xenografts

A

human tissue is placed in the mouse brain

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10
Q

Xenografts Strengths and Weaknesses

A

Strengths: able to study live human neurons and manipulate experimentally
Weaknesses: growth might be different due to neurons being dissociated or growing in mice

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11
Q

Main requirements for Anatomy and Physiology

A

Anatomy: whole intact tissue (not in a dish)
Physiology: live cells

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12
Q

Ways to study whole intact tissue

A

Blocking slabs, dissecting - human hippocampus example

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13
Q

What does histology allow you to see?

A

human hippocampus stained

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14
Q

What are different types of histology?

A

Common structural stains: H&E
Hematoxalin: (Blue - stains nuclei)
Eosin: (pink - stains cytosol and extracellular matrix)
Pathology standard

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15
Q

What is a common structural stain for histology?

A

Nissl stains - multiple dyes reveal Nissl substance
Nissl: bound ribosomes and DNA

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16
Q

What is another common structural stain for histology?

A

Golgi stain
- random fills 1 in 10,000 cells
Ex: human hippocampus

17
Q

When doing histology, what makes cells unique/distinguishable from one another?

A
  1. gene protein/expression
  2. shape/size (morphology)
  3. physiological properties
  4. anatomical location/circuit connectivity
  5. composition of intracellular components
18
Q

Histology - mRNA detection definition

A

reveals cells expressing a specific mRNA transcript

Strengths: can look at any genes high resolutions
Weaknesses: technically challenging, dead tissue

19
Q

Histology - protein detection

A

through immunohistochemistry vs. immunofluorescence

Ex: double cortin (Dcx) protein expressed by immature neurons

Strengths: can look at the localization of many proteins at high resolution
Weaknesses: many caveats/non-specific staining/ protein degradation, dead tissue

20
Q

Antibody Labeling (Primary vs Secondary)

A

Primary Antibodies:
- recognize protein of interest (ex. DCX)
- are generated in different host species (ex: rabbit)

Secondary Antibodies:
- recognize antibodies from a specific host
- are generated in different host species
- are directly linked to enzymes or fluorescent molecules to reveal their location

21
Q

Physiology approach

A

recording electrical currents from living neurons: properties change as neurons develop

22
Q

Pharmacology approach

A

administer a drug and evaluate response to changing doeses or time
- agonists/ antagonists
Strengths: repeated-measures over time, titration curves
Weaknesses: off-topic effects, non-physiological

23
Q

What are the two types of genetic knock out mice?

A

Global knockout
- animal doesn’t have the gene ever
- advantage: sledgehammer
- disadvantage: sledghammer

Conditional knockout
- animal has the gene until some other factor causes it to be actively eliminated
Advantage: much more selective
Disadvantage: effects may be subtle

24
Q

What are development-focused techniques?

A

Birthdating: labelling replicating DNA during cell divisions:

H-Thymidine or BrdU
- Thymidine analogs look like Thymidine, become incorporated into newly-born cells
Advantage: labels dividing cells, easy to be co-label with other cell markers
Disadvantage: somewhat toxic, label can be diluted with subsequent divisions

Carbon Dating
- measure ratio of C14 to C13 in cells to determine if it corresponds to biosphere levels at the age of the animal
Advantage: possible to birthdate in postmortem tissue
Disadvantage: prone to contamination/ other sources of C / DNA repair/methylation

25
Q

What is another development-focused technique?

A

Fate Mapping: following the fate of a cell over time
Requirements:
1. clear identification of initially labeled cells
2. label does not diffuse into neighboring cells
3. label is stable and non-toxic

26
Q

What are some ways to fate map?

A

Dyes: Walter Vogt
- Electroporation
-Viruses
-Transplantation
-Time-lapse imaging

27
Q

Vital Dyes

A

Lipophilic Dyes: rapidly incorporated into phospholipid bilayer

28
Q

What are the advantages and disadvantages of lipophilic dyes?

A

Advantages: rapid labeling in live or dead tissue
- labels entire cell structure, can be costained with cell type markers

Disadvantages: non-selective, diffuses across large area, filling many cells

29
Q

Transplantation

A

powerful technique, used frequently in developmental neuroscience
- dissect tissue from one area or animal
- place in an ectopic area or animal

30
Q

What does transplantation reveal?

A

-reveals patterning
-can test cell autonomy vs. non-autonomy
-makes chimeric animals (genetic or heterochronic)
Xenografts - transplantation across species

31
Q

Fate Mapping: time-lapse imaging

A

-making a movie from repeated images of the same tissue
-requires live cells
- ideally in the animal directly
-slice cultures of brains are next best
-dissociated cells or tissue culture depending on the question

Movie: viral labeling, transplantation, time-lapse imaging

32
Q

What is single cell lineage tracing?

A

C. elegans/D. rerio

33
Q

What is single cell (or nuclei) RNA sequencing

A

scRNA-seq

34
Q

What is the strength and weakness of lineage tracing?

A

S: can infer dynamics in dead tissue
W: lack of anatomy

35
Q

What does it mean to be Necessary?

A

absence of A guarantees absence of B
- genetic knockouts, laser or chemical ablations

36
Q

What does it mean to be Sufficient?

A

presence of A guarantees presence of B
- genetic over-expression, transplantation experiments

37
Q

Correlation Example

A

Do neural stem cells generate neurons?
- We see them present in the same location, but how do we learn about causation?

38
Q

LECTURE 1 TAKE HOME MESSAGE

A

many experimental techniques in combination help buiild evidence for how the brain is built. careful observation is the first step, followed by experimentation to determine necessity and sufficiency

Developmental Neuro (2 decks)

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