Genetic Tools for Studying Neural Circuit Function:

Question 1

Briefly outline neural circuits and tactics to achieving subtype specificity

Answer 1

• There are lots of cell types in the CNS, all doing different things
• This makes manipulating specific cell types in a spatially-discrete manner difficult on face value
o But, different cell types express different genes:
 All cells express synapsin (Syn)
 Only glutamatergic neurons express Ca2+/calmodulin-dependent protein kinase 2a (CamK2a)
 Only cortical and hippocampal pyramidal cells express Emx1
 Only inhibitory interneurons express glutamic acid decarboxylase (GAD1/2)
 Only dopaminergic neurons express DAT
 Only serotonergic neurons express SERT
o Thus genetics allows us to achieve subtype specificity
o It’s possible to use a cell-subtype specific promotor to target cells for manipulation
 Knowing the promoter sequence of any gene allows the downstream insertion of any genetic tool e.g. GFP, TdTomato

Question 2

Explain the Cre-LoxP system and its applications

Answer 2

• Creating new animals which express genes under a specific promoter costs money and time
• The Cre-LoxP system is a flexible system which allows the introduction of new genes, or the inactivation of existing ones
o Cre recombinase targets short nucleotide sequences (LoxP) for recombination
o This system is derived from the bacterial immune system
• Can be used to turn genes on or off
• Using this requires 2 things:
o Cre-recombinase expression driven by a specific promoter
o Gene/genetic tool of interest, surrounded by Lox sites
• Promoters:
o hSyn: human synapsin; a ubiquitous neuronal promoter
o CamK2a: Ca2+/calmodulin-dependent protein kinase 2a – expressed in glutamatergic neurons
o Any other gene – GFAP, PV, DAT, SERT, SOM
• Genetic tools:
o Fluorescent proteins – GFP
o Optogenetic tools – ChR2
o Artificial drugs/receptors (DREADDS)
o Any protein you want to specifically express or delete

Question 3

Name 3 conditional expression systems and state their mechanism of action

Answer 3

1. Cre-LoxP system:
   o Uses Cre recombinase to invert a sequence of DNA surrounded by Lox sites
2. Tet on/off system:
   o Tetracycline-controlled transcriptional activation
   o Giving tetracycline (an antibiotic) can either turn on or turn off the expression of specific genes
3. FLP-FRT system:
   o Similar to Cre-LoxP
   o Flippase (FLP) recombinase recognises a pair of FLP recombinase target (FRT) sequences which flank a genomic region of interest

Question 4

Outline viral transfection systems and the types of viruses and their considerations

Answer 4

• Viral transfection  expression of transgene
o Typically use a lentivirus or adenoassociated virus (AAV)
• Can:
o Inject virus to transduce transgene driven by a specific promoter contained within the virus
o Inject the virus to transduce a floxed transgene into a Cre-mouse

•	Choice of virus is dictated by the experimental requirements
o	‘payload’ size
o	Speed of expression
o	Safety
o	Availability ‘off-the-shelf’

Virus type: Maximum insert size: Considerations:
Adenoassociated virus (AAV) <4kb Slow expression onset
Lentivirus 8kb Derived from HIV, inserts into host genome
Herpes simplex >30kb Large insert capacity but often used
Rabies virus <4kb Retrograde infection – kills the host cells within 14 days

Question 5

Explain the advantages and disadvantages of using viruses to study neural circuits

Answer 5

Virus pros (vs mice):
Don’t necessarily need a transgenic animal; can express in any animal
Faster breeding than in mice
Spatial selectivity – can target just 1 brain region

Virus cons (vs mice):
Limited to the size of the payload that you can fit inside the viral genome (some promoter sequences are just too large for viruses to contain)
No control over where into the genome your transgenes get expressed
Variability in expression levels
Biological safety issues

Question 6

Give an overview of in utero electroporation and examples of its applications

Answer 6

In utero electroporation:
• Use of viral vectors requires 2-3 weeks to achieve useful transgene expression
o This isn’t much use for studying early developmental timepoints
• In contrast, in utero electroporation can be used in mice from E12.5
o Inject DNA then use electric shock to cause uptake and integration; expression then occurs as normal
• Can use this to study schizophrenia and autism spectrum disorders

Examples:
• Conditional KOs:
1. surround gene of interest by LoxP sites
2. Use a Cre driver mouse to delete the gene from specific cell types
1. Delete Tsc1 from some interneurons is sufficient to reduce birth weight and decrease survival in mice
a. Tsc1 is part of the mTOR pathway, this is important for development
• Cell-specific silencing:
o Tetanus light-chain toxin (TeLC):
 Blocks vesicle docking on presynaptic membrane
 Silences neurons without killing them
 E.g. parvalbumin (PV) neurons are required for spatial working but not spatial reference/memory, using a radial arm maze/Morris Water Maze
• Path length shortens = mice learning escape location
• More incorrect entries = mice can’t remember which arms they’ve visited
• Inducible knock-in:
o Tamoxifen-inducible Cre (CreER):
 Cre recombinase is active only when tamoxifen is present in plasma
 Tamoxifen crosses the placental barrier; CreER is active for ~1 day
 Can use this to birth-date neurons

Question 7

Outline optogenetics, including its advantages and disadvantages

Answer 7

• Can use light-gated ion channels to control neuronal activity
• These can be excitatory (ChR2) or inhibitory (halorhodopsin or archaerhodopsin)
o ChR2: cation channel (excitatory, Na+, K+)
o NpHR: chloride pump (inhibitory)
o ArchT: proton pump (inhibitory)
• Express these in cells using genetic methods: transgenic mice/viruses
o Combine this with electrophysiology or behaviour to interrogate neuron function
• Can use this to silence or activate cells e.g. Basting and neurogenic hypertension

Pros:
Use of optically-excitable ion channels allows rapid control of neuronal excitability
Allows cell-specific (using Cre-LoxP) and/or region-specific (using viral injection) control of neuronal pathways

Cons:
Need a light source to activate the opsin (tricky for in vivo behavioural experiments)
Potential for ion channels to perturb normal neuron physiology (in practice this is only really a concern with halorhodopsin)

Question 8

Outline chemogenetics, including its advantages and disadvantages

Answer 8

• AKA pharmacogenetics
• Involves the use of DREADDs:
o Designer Receptors Exclusively Activated by Designer Drugs
 Modified neurotransmitter receptors that don’t respond to endogenous ligands
 Activated by ‘designer’ compounds
• Can be delivered in the same way as any other genetic tool, e.g. a viral vector, transgenic mouse, with/without Cre-LoxP
• First developed by Bryan Roth:
o hM3Dq – modified M3 muscarinic receptor – excitatory GPCR
o hM4Di – modified M4 muscarinic receptor – inhibitory GPCR
o Activated by ligands such as clozapine-N-oxide (CNO) or agonist 21

Pros:
No light source needed
Fully reversible effects – just don’t give the agonist!
Fairly cheap setup vs optogenetics

Cons:
Less temporal control than optogenetics for activation (GPCR vs ion channel)
Specificity of ‘inactive’ agonists – CNO
Lots of controls needed: DREADD with ligand/vehicle, control with ligand/vehicle. If combining with transgenic disease model this rapidly increases the number of groups