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

Oct 07, 2023 •359 likes •766 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

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

  2. Gaining experimental access to neural circuits

  3. Adeno-associated virus (AAV)

  4. Adeno-associated virus (AAV)

  5. Adeno-associated virus (AAV)

  6. Recombinant AAV (rAAV) 4.9 Kb

  7. rAAV – Targeting the right cell type – how?

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

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

  10. rAAV capsid tropism – natural variants

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

  12. rAAV capsid tropism – engineered variants

  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

  14. rAAV – selective promoters ~4.9 Kb

  15. rAAV – recombinase expression Atasoy et al., 2008

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

  17. Intersectional targeting with AAVs ChAT CGRP Victoria Tung

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

  19. Defining inputs to genetically identified neurons

  20. Rabies virus as a retrograde transsynaptic tracer

  21. Rabies virus as a retrograde transsynaptic tracer

  22. Monosynaptic tracing with rabies virus Schnell et al., 2010

  23. Transsynaptic tracing with rabies virus glycoprotein complementation

  24. Transsynaptic tracing with rabies virus

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

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

  27. Producing rabies vectors in the lab

  28. EnvA-pseudotyping of rabies virus

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

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

  31. Disadvantages of rabies tracing Toxicity Low transsynaptic spread

  32. Different strains of rabies virus have different properties

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

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

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

  36. Identifying direct and indirect inputs to LVN-spinal neurons

  37. Identifying direct and indirect inputs to LVN-spinal neurons

  38. Identifying direct and indirect inputs to LVN-spinal neurons

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