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NEUR0020 > Techniques in Functional Neuroanatomy > Flashcards

Techniques in Functional Neuroanatomy Flashcards

1
Q

Name 2 methods to identify neuron outputs

A

Retrograde tracing

Transynaptic retrograde viral tracing

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2
Q

Name 2 methods to identify neuron inputs

A

Anterograde tracing

Lesion neurons/axons

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3
Q

Name 5 methods to test the functionality of connections

A

C-fos
Electrophysiology - stimulation and recording
Calcium imaging
Optogenetics
Genetic manipulation of targeted neurons - ablation/silencing

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4
Q

What are the 2 main transgene insertion methods?

A

Genetic targeting

Viral vector targeting

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5
Q

What are the 3 genetic targeting approaches to transgene insertion?

A

Transgenic approach
Binary expression systems
Inducible systems

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6
Q

What are the 2 types of binary expression system?

A

Recombination-based - Cre-loxP

Transactivation-based - tTa-tetO

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7
Q

What are the 3 viral vector targeting approaches to transgene insertion?

A

Viral constructs
Engineered tropism
Axon transduction

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8
Q

Which 2 types of transgenes can be inserted for neural circuit studies?

A

Optogenetics

Chemogenetics - DREADDs

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9
Q

What are the components of a transgene cassette?

A

Exogenous gene
With/without promoter
Recombination sites

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10
Q

What are the 2 methods used in the transgenic approach?

A

Conventional - transgene cassette

BAC

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11
Q

What are the advantages of BAC over transgene cassette?

A

BAC is large - longer transgene

Can contain more regulatory elements - more complex expression regulation

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12
Q

What is the driver line in binary expression systems?

A

Expresses driver gene (e.g. Cre)

Controlled specific promoter - gives cell specificity

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13
Q

What is the reporter line in binary expression systems?

A

Expresses transgene of interest

Controlled by robust ubiquitous promoter - gives high expression levels

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14
Q

Which promoter is specific for excitatory neurons?

A

CAMKII

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15
Q

Which promoter is specific for inhibitory neurons?

A

NPY

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16
Q

How is Cre-lox used to cause a selective transgene deletion?

A

LoxP sites flank transgene - within promoters

Cre recombination - transgene deletion only where Cre expressed

17
Q

How is Cre-lox used to cause a selective transgene knock-in?

A

Stop codon flanked by loxP sites - lox stop lox (LSL) - precedes transgene
Cre recombination - deletion of stop codon - transgene expressed only where Cre expressed

18
Q

How is Cre-lox used to selectively convert a transgene from inactive to active?

A

FLEX-switch system
Transgene in antisense orientation - flanked by 2 pairs of loxP sites
Cre recombination - transgene flipped to sense orientation only where Cre expressed - transgene expressed

19
Q

What is a benefit of the FLEX-switch system?

A

Can use 2 transgenes in cassette - opposite orientations - where Cre expressed switch one on and other off

20
Q

How does CreER work?

A

CreER - Cre + ligand-binding domain of oestrogen receptor
CreER in cytoplasm
Add tamoxifen (oestrogen analogue) - Cre translocates to nucleus - acts on loxP sites
Cre active on-demand

21
Q

What is tTA-tetO?

A

Transgene expressed in absence of tetracycline/doxycycline

Reversible

22
Q

How does a viral construct work?

A

Insert transgene into viral vector
Vector infects host cells
Different virus types integrate/do not integrate into host DNA
Transgene transcribed

23
Q

What is engineered tropism and what is its benefit?

A

Viral vector coated with protein - only infects cells expressing receptor for protein (TVA)
Enables host cell type-specific transgene expression

24
Q

How does axon transduction work?

A

Transgene in recombinant rabies virus deficient in glycoprotein - infects axon terminals - cannot cross synapse
Reintroduce glycoprotein - virus crosses synapse - infects other cells

25
Q

What is a disadvantage of axon transduction?

A

High cytotoxicity

26
Q

Name 3 main viral vectors?

A

Recombinant adeno-associated viral vector
Retroviral vector
Gutless adenoviral vector

27
Q

At what timescale does optogenetics allow the control of neuronal activity?

A

Millisecond

28
Q

How can opsin expression be visualised?

A

Insert gene for reporter molecule after opsin gene in transgene cassette - all under cell-specific promoter

29
Q

Name 2 reporter molecules

A

mCherry

tdTomato

30
Q

What are DREADDs?

A

Designer receptors exclusively activated by designer drugs

31
Q

Which DREADD is a Gq-coupled receptor, which ligand activates it, and what is its effect on neuronal activity?

A

hM3D
Activated by CNO
Activates neuron

32
Q

Which DREADD is a Gi-coupled receptor, which ligand activates it, and what is its effect on neuronal activity?

A

hM4D
Activated by CNO
Silences neuron

33
Q

What is a disadvantage of DREADDs?

A

Same agonist (CNO) activates hM3D and hM4D - cannot simulatenously express both in same neuron to switch on/off

NEUR0020 (11 decks)

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