Viral vectors for neural circuit analysis Andy Murray - - PowerPoint PPT Presentation

▶
viral vectors for neural circuit analysis
SMART_READER_LITE
LIVE PREVIEW

Viral vectors for neural circuit analysis Andy Murray - - PowerPoint PPT Presentation

Oct 07, 2023 359 likes •767 views

Viral vectors for neural circuit analysis Andy Murray a.murray@ucl.ac.uk Gaining experimental access to neural circuits Adeno-associated virus (AAV) Adeno-associated virus (AAV) Adeno-associated virus (AAV) Recombinant AAV (rAAV) 4.9 Kb

slide-1
SLIDE 1

Andy Murray a.murray@ucl.ac.uk

Viral vectors for neural circuit analysis

slide-2
SLIDE 2

Gaining experimental access to neural circuits

slide-3
SLIDE 3

Adeno-associated virus (AAV)

slide-4
SLIDE 4

Adeno-associated virus (AAV)

slide-5
SLIDE 5

Adeno-associated virus (AAV)

slide-6
SLIDE 6

Recombinant AAV (rAAV)

4.9 Kb

slide-7
SLIDE 7

rAAV – Targeting the right cell type – how?

slide-8
SLIDE 8

How do we target specific neuron types with AAV?

  • 1. capsid tropism
  • 2. selective promotor
  • 3. recombinase expression
slide-9
SLIDE 9

rAAV capsid tropism – natural variants

Vance et al., 2015

slide-10
SLIDE 10

rAAV capsid tropism – natural variants

slide-11
SLIDE 11

rAAV capsid tropism – engineered variants

http://2010.igem.org/Team:Heidelberg/Notebook/Capsid_Shuffling/Homology_Based

slide-12
SLIDE 12

rAAV capsid tropism – engineered variants

slide-13
SLIDE 13

rAAV capsid tropism – engineered variants

AAV-DJ (Grimm et al., J. Virol. 2008)

  • Broad neuronal tropism

AAV-Retro (Tervo et al., Neuron. 2016)

  • Infects neuronal terminals

AAV-Php.eB (Deverman et al., Nat. Biotech. 2016)

  • Infects CNS after venous injection

AAV-PhP.S (Deverman et al., Nat. Biotech. 2016)

  • Infects PNS after venous injection
slide-14
SLIDE 14

rAAV – selective promoters

~4.9 Kb

slide-15
SLIDE 15

rAAV – recombinase expression

Atasoy et al., 2008

slide-16
SLIDE 16

How do we target specific neuron types with AAV?

promoter capsid tropism recombinase expression

slide-17
SLIDE 17

Intersectional targeting with AAVs

CGRP ChAT

Victoria Tung

slide-18
SLIDE 18

Victoria Tung

CGRP promoter GCaMP pA ITR ITR

Intersectional targeting with AAVs

ChAT::Cre; tdTomato

slide-19
SLIDE 19

Defining inputs to genetically identified neurons

slide-20
SLIDE 20

Rabies virus as a retrograde transsynaptic tracer

slide-21
SLIDE 21

Rabies virus as a retrograde transsynaptic tracer

slide-22
SLIDE 22

Monosynaptic tracing with rabies virus

Schnell et al., 2010

slide-23
SLIDE 23

Transsynaptic tracing with rabies virus

glycoprotein complementation

slide-24
SLIDE 24

Transsynaptic tracing with rabies virus

slide-25
SLIDE 25

Transsynaptic tracing with rabies virus

targeting initial infection with EnvA/TVA

slide-26
SLIDE 26

Transsynaptic tracing with rabies virus

targeting initial infection with EnvA/TVA

slide-27
SLIDE 27

Producing rabies vectors in the lab

slide-28
SLIDE 28

EnvA-pseudotyping of rabies virus

slide-29
SLIDE 29

Watabe-Uchida et al., 2012

Circuit mapping with rabies virus

slide-30
SLIDE 30

Watabe-Uchida et al., 2012

Circuit mapping with rabies virus

slide-31
SLIDE 31

Disadvantages of rabies tracing

Toxicity Low transsynaptic spread

slide-32
SLIDE 32

Different strains of rabies virus have different properties

slide-33
SLIDE 33

Monitoring neural activity with rabies

Experimental design Measuring calcium transients in hippocampus

slide-34
SLIDE 34

Channelrhodopsin stimulation with N2c rabies virus

Experimental design Photostimulation Generation of postsynaptic potentials

slide-35
SLIDE 35

Nissl Fluorogold (lumbar cord)

Identifying monosynaptic inputs to LVN-spinal neurons

Genetic marker Projection pattern Stereotaxic coordinates

slide-36
SLIDE 36

Identifying direct and indirect inputs to LVN-spinal neurons

slide-37
SLIDE 37

Identifying direct and indirect inputs to LVN-spinal neurons

slide-38
SLIDE 38

Identifying direct and indirect inputs to LVN-spinal neurons

slide-39
SLIDE 39
slide-40
SLIDE 40