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

Viral vectors for neural circuit analysis Andy Murray a.murray@ucl.ac.uk

Gaining experimental access to neural circuits

Adeno-associated virus (AAV)

Recombinant AAV (rAAV) 4.9 Kb

rAAV – Targeting the right cell type – how?

How do we target specific neuron types with AAV? 1. capsid tropism 2. selective promotor 3. recombinase expression

rAAV capsid tropism – natural variants Vance et al., 2015

rAAV capsid tropism – natural variants

rAAV capsid tropism – engineered variants http://2010.igem.org/Team:Heidelberg/Notebook/Capsid_Shuffling/Homology_Based

rAAV capsid tropism – engineered variants

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

rAAV – selective promoters ~4.9 Kb

rAAV – recombinase expression Atasoy et al., 2008

How do we target specific neuron types with AAV? promoter capsid tropism recombinase expression

Intersectional targeting with AAVs ChAT CGRP Victoria Tung

Intersectional targeting with AAVs ChAT::Cre; tdTomato GCaMP ITR CGRP promoter pA ITR Victoria Tung

Defining inputs to genetically identified neurons

Rabies virus as a retrograde transsynaptic tracer

Monosynaptic tracing with rabies virus Schnell et al., 2010

Transsynaptic tracing with rabies virus glycoprotein complementation

Transsynaptic tracing with rabies virus

Transsynaptic tracing with rabies virus targeting initial infection with EnvA/TVA

Producing rabies vectors in the lab

EnvA-pseudotyping of rabies virus

Circuit mapping with rabies virus Watabe-Uchida et al., 2012

Disadvantages of rabies tracing Toxicity Low transsynaptic spread

Different strains of rabies virus have different properties

Monitoring neural activity with rabies Experimental design Measuring calcium transients in hippocampus

Channelrhodopsin stimulation with N2c rabies virus Photostimulation Experimental design Generation of postsynaptic potentials

Identifying monosynaptic inputs to LVN-spinal neurons Nissl Fluorogold (lumbar cord) Genetic marker Projection pattern Stereotaxic coordinates

Identifying direct and indirect inputs to LVN-spinal neurons

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

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

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