Rabies Virus

B19G2

5 Genes

 

RVG is required for transsynaptic spread, but not transcription or viral genome replication.

(Etessami et al., 2000, Mebatsion et al., 1996 and Wickersham et al., 2007) 

Cell type specific delivery

Applications and limitations

1. sparse labeling(a few cells)

Cell type?

2. sparse/intensive labeling

  a) promotor too long/ unknown promotors

  b)DIO/FLEX-TVA-mCherry

+DIO/FLEX-RVG

3. Retrograde tracing

Hard to control virus spreading

EnvA-TVA()

Delivering TVA and RVG into starting cells

Different ways:

1.Electroporation

2.Virus(AAV/lenti virus)

  a) Promotor

  CamKIIa, Somatostatin, Ef1a...

  b)CRE animal

  DUAL VECTORS SYSTEM

3. Transgenic animal

  CRE>>TVA+RVG

Problems with

DUAL VECTORS SYSTEM

Starter cell:

TVA-mCherry+, RVG+, Rabies GFP+

 

TVA-mCherry+, RVG-, Rabies GFP+

=> Overestimate the number of starting populations

=> Not possible to do local input dissection(e.g., between layers in the cortex...)

 

Solutions

1. Tag RVG

=>sensitive to tags (personal contact with Ed Callaway)

2. All in one vector

EF1a-DIO-TVA-mCherry-t2A-RVG

=>too large 5-6 kilo basepairs

3.EF1a-DIO-TVA-t2A-RVG

=>Need to stain for different antibodies to see starter cells(e.g., unstable proteins like PV)

4.SINGLE VECTOR rabies system

EF1a-DIO-TVA-V5-t2A-RVG

SINGLE VECTOR rabies system

• V5
• Paramyxovirus SV5

• a small epitope present on the P and V proteins of SV5

• 14 aa=>42bp

• works good in AAV vectors
• Fusion protein with TVA on C-terminal=>membrane bound

double virus system

Rabies Virus

• ChR2-functional study
• other opsins
• other modulators
• disadvantage
• limited time window

3'-N-P-M-GFP-L-5'

 

3'-N-P-M-ChR2-mCherry-L-5'

3'-N-P-M-SSFO-EYFP-L-5'

3'-N-P-M-Jaws-GFP-L-5'

Prefrontal Cortex

• why？
•  

WholeBrain

 Whole brain inputs to PV and SOM neurons in prefrontal cortex

Globus Pallidus

Median Septum

Basolateral Amygdala

Ventral group of the dorsal thalamus

Hippocampus-CA1,Stratum Oriens

Local Inputs

Summary

• Prefrontal cortex interneurons(PV and SOM) get more local inputs than long-range inputs.
• Prefrontal cortex interneurons(PV and SOM) get inputs mainly from cortical areas.
• Prefrontal cortex interneurons(PV and SOM) also get inputs from Globus Pallidus(Cholinergic), Amygdala, ventral group of the dorsal thalamus and Hippocampus.
• No obvious differences found in between genotypes.

On-going work

• Tracing project:
• Other interneurons (VIP)
• Principal cells (Coinjection with CaMKIIa-CRE)
• New vector designs: CaMKIIa-TVA-V5-t2A-RG

Acknowledgements

• Marie Carlén
• Konstantinos Meletis

• Sofie Ährlund-Richter
• Daniel Fürth

• Xinming Wang
• Calvin Young

• Other members in the lab

Thank you!

    Advantages and Disadvantages

TVA-V5-RG Vs TVA-mCherry+RG(TVA-mCherry/non-RG infected cells)

Local input

•Disadvantages:

Staining

Alternative:
  AAV8/synP-FLEX-sTpEpB

  (Keigo Kohara et al., Nature Neuroscience 2014)
  Reduction of total genome length by:
    using smaller promotor(human synapsin-1 promotor)

    using smaller polyadenylation signal(bovine growth hormone polyadenylation site)
    removing all extraneous sequences and restriction sites
    7 days+ 7 days