Techniques in Functional Neuroanatomy

Question 1

Name 2 methods to identify neuron outputs

Answer 1

Retrograde tracing

Transynaptic retrograde viral tracing

Question 2

Name 2 methods to identify neuron inputs

Answer 2

Anterograde tracing

Lesion neurons/axons

Question 3

Name 5 methods to test the functionality of connections

Answer 3

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

Question 4

What are the 2 main transgene insertion methods?

Answer 4

Genetic targeting

Viral vector targeting

Question 5

What are the 3 genetic targeting approaches to transgene insertion?

Answer 5

Transgenic approach
Binary expression systems
Inducible systems

Question 6

What are the 2 types of binary expression system?

Answer 6

Recombination-based - Cre-loxP

Transactivation-based - tTa-tetO

Question 7

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

Answer 7

Viral constructs
Engineered tropism
Axon transduction

Question 8

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

Answer 8

Optogenetics

Chemogenetics - DREADDs

Question 9

What are the components of a transgene cassette?

Answer 9

Exogenous gene
With/without promoter
Recombination sites

Question 10

What are the 2 methods used in the transgenic approach?

Answer 10

Conventional - transgene cassette

BAC

Question 11

What are the advantages of BAC over transgene cassette?

Answer 11

BAC is large - longer transgene

Can contain more regulatory elements - more complex expression regulation

Question 12

What is the driver line in binary expression systems?

Answer 12

Expresses driver gene (e.g. Cre)

Controlled specific promoter - gives cell specificity

Question 13

What is the reporter line in binary expression systems?

Answer 13

Expresses transgene of interest

Controlled by robust ubiquitous promoter - gives high expression levels

Question 14

Which promoter is specific for excitatory neurons?

Answer 14

CAMKII

Question 15

Which promoter is specific for inhibitory neurons?

Answer 15

NPY

Question 16

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

Answer 16

LoxP sites flank transgene - within promoters

Cre recombination - transgene deletion only where Cre expressed

Question 17

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

Answer 17

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

Question 18

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

Answer 18

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

Question 19

What is a benefit of the FLEX-switch system?

Answer 19

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

Question 20

How does CreER work?

Answer 20

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

Question 21

What is tTA-tetO?

Answer 21

Transgene expressed in absence of tetracycline/doxycycline

Reversible

Question 22

How does a viral construct work?

Answer 22

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

Question 23

What is engineered tropism and what is its benefit?

Answer 23

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

Question 24

How does axon transduction work?

Answer 24

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

Question 25

What is a disadvantage of axon transduction?

Answer 25

High cytotoxicity

Question 26

Name 3 main viral vectors?

Answer 26

Recombinant adeno-associated viral vector Retroviral vector Gutless adenoviral vector

Question 27

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

Answer 27

Millisecond

Question 28

How can opsin expression be visualised?

Answer 28

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

Question 29

Name 2 reporter molecules

Answer 29

mCherry | tdTomato

Question 30

What are DREADDs?

Answer 30

Designer receptors exclusively activated by designer drugs

Question 31

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

Answer 31

hM3D Activated by CNO Activates neuron

Question 32

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

Answer 32

hM4D Activated by CNO Silences neuron

Question 33

What is a disadvantage of DREADDs?

Answer 33

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