CALLAWAY, Ed  Rabies Virus for Elaborating Circuit Structure, Salk, Sept 27 2013

title:  linking circuit structure and function with G-deleted rabies virus:
 prospects for anterograde and for orthogonal circuit tracing...

into by Karl Deisseroth;

some of these tools are still on the drawing board  (since we at Stfd are early tool adopters)

want rabies virusto go anterograde (to the POST-synap neuron; normally into just
goes retrograde to presynap neuron) ...

OUTLINE:

we need tools that allow direct causal and correl studies linking neural circuitry to function...

neural connections are cell-type specific...

not nec related just to physical overlap in space 
 (cuz  axons can skip over close by neurons in a cell-type specific manner)

must be able to link circuit and function in partic cell types...

rabies virus does not kill cells immediately... 
so u have window where u can do function studies...

********************

see Wickersham in Neuron 2007;

identif neurons that are directly presynap to a single neuron
 or to an entire genetically targeted population

new technique: now taking off:  using transcomplemented rabies tech..

glyoprotein deleted rabies virus...

he shows rat cortex  with green GFP...

initial cell  (one cell infec by rabies) which then spreads retrograde presynaptically...  
(from  wickersham 2007)

so, how did we target just ONE cell  (unlike wild type rabies)  and
 how did we do it so that it goes only ONE step backward.
(this is a review of this very famous work that he showed at Stanford several months
ago during a previous talk. qv)

glycoprotein deletion is the key.

rabies virus is coated by glycoprotein
delete that and replace with some other gene/ protein.

alter the host cell to have that  glycoprotein in specific cells...

inject into barrel cortex of mouse... 
that labels pyramidals and their apical dendrites  (layer 2/3 pyrams)

use single cell electroporation
use TVA, rabies G and YFP..
see Marshel in Neuron 2010...

express mCherry in one step retrograde neurons...
 great slide with initial target cell and presynap cells...
you can then visualize 300 to 400 inputs to cell...

next...  use Cre-dependent cells...  see Wall in PNAS 2010

only Cre+ cells will express TVA...

in the absence of the glycoprot  rabies does not spread.

cerebellum:  can label population of cells;
****************************************(above is his (v famous) standard work)

can put arbitrary genes into rabies to monitor activity of neuron, etc.  
eg  to monitor  GCaMP3 or 6...

can use to express ChR2 = channel rhodopsin...

********************  now,  how to get rabies to  go antegrade!  (thot to be impossible)

anterograde transsynap tracing

rabies virus only spreads retrogradely

herpes  HSV and PRV  goes both way but the H129  strain of HSV only goes anterograde...

but HSV kills cells and system does not just go monosynaptically...

but looked at VSV = stomatitis virus... but perhaps
 can take  glycoproteins from HSV H129 to make rabies go anterograde...

shoot 11 plasmids! into gene gun...

HSV glycoproteins CAN mediate  anterograde spread...

work by Al Cetin...

is the spread antero or retro or both?
he also expressed ChR2 in starter cell...

starter cells in yellow;  then photoactivate  the yellow cells..

Jump is monosynap and in the anterograde direction.
put on TTX  = tetrodotoxin... (to control it)

*******************  last topic

"orthogonal" transynap tracing...

want to trace two separ starter cells (to keep 2 cells presynaptically cells separate even if
they are connected)  do this with Sensai virus...

Q  does rabies virus alwas kill the neuron  ?  prob yes... we are trying v hard to prevent that..
after 2 weeks almost all cells are dead (despite our best efforts)......
virus wants to spread  (but there is innate immunity  but virus can blunt immune response
 in order to spread more)

Q  are you labelling all the cells that are presynap?  NO.. perhaps about 10%
might be getting close to 100%