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@as_bates | |||||
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We then tried a challenging test. Third-order ‘LHNs’ integrate stereotyped combinations of 5+ input glomeruli. #BAcTrace revealed 12/16 connections expected from EM #connectomics data with a 10 synapse threshold. The top partner had >400 outputs onto the donor cells. 6/11 pic.twitter.com/aeSGhvrzLf
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Greg Jefferis
@gsxej
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29. sij |
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#BAcTrace, a #tweeprint: At least since Francis Crick’s famous review of 1979, neuroscientists have dreamed of ways to identify all the neurons connected to a cell of interest. In the last decade this has become a reality through the widespread use of rabies virus tracers. 1/11 twitter.com/biorxivpreprin… pic.twitter.com/nVGKPvCexH
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Greg Jefferis
@gsxej
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29. sij |
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Rabies has been engineered to act as a retrograde tracer, revealing all the inputs to a neuron. However limitations include toxicity and uncertainty about which synapses it crosses. Furthermore, these viruses are not applicable to all species. 2/11
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Seba
@SebaCachero
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29. sij |
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We have developed a new fully genetically encoded tracing system, #BAcTrace, and validated it in #Drosophila. This makes the fly the first animal with such systems for both retrograde and anterograde tracing (trans-Tango, TRACT). 3/11 pic.twitter.com/p6i4Qav2lI
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Seba
@SebaCachero
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29. sij |
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In #BAcTrace one genetic driver defines a *donor* population expressing an engineered C. botulinum neurotoxin (aka #botox!). This jumps backwards across the synapse to *receiver* neurons. The botox then releases a tethered transcription factor in the connected cells. 4/11 pic.twitter.com/dn7XZ7SeSW
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Seba
@SebaCachero
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29. sij |
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We first validated #BAcTrace in two well characterised connections in the fly olfactory system. One was weak but stereotyped and highly selective (ORN-PN), the other is strong but characterised by random convergence of multiple cell types onto downstream neurons (PN-KC). 5/11 pic.twitter.com/q56e5gARcD
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Shahar
@sfrechter
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29. sij |
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Although botox is a powerful toxin, we have engineered #BAcTrace to be non-toxic to flies (and humans). This allowed us to make whole cell patch clamp recordings from connected neurons for at least 1-2 weeks after BAcTrace labelling. 7/11 pic.twitter.com/5YpvwC3ZkG
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Seba
@SebaCachero
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29. sij |
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After almost 10y #BAcTrace works well, but 1) you need a LexA driver for your receiver (upstream) neurons 2) high sensitivity means you will get a BAcTrace signal in neurons that express both the receiver and donor constructs – even if the overlap is at very low levels. 8/11
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