Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus

J. Patrick Card, Oren Kobiler, Joshua McCambridge, Sommer Ebdlahad, Zhiying Shan, Mohan K. Raizada, Alan F. Sved, and Lynn W. Enquist. Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus. Proc Natl Acad Sci U S A. 108 (8): 3377-82, 2011.

My first blog, yeah!! 

     This paper that is based on a collaboration between Alan Sved, Mohan Raizada, and Lynn Enquist.  We've had previous blogs on papers by Alan Sved, a leader in our field for about 20 years.  This paper in particular highlights Alan Sved's interest in tracing the neural networks involved in blood pressure control.  In this paper they use a virus to trace in a retrograde fashion the sympathetic pathways the control the kidney.  This is accomplished by injecting the virus into the kidney, which then travels back up the sympathetic postganglionic nerves (hence retrograde tracing) to the sympathetic ganglion.  Here in the ganglion the virus crosses the synapse (a.k.a. "goes transynaptic") to infect and replicate in the terminals of the preganglionic nerves that innvervate the post-ganglionic nerves that innervate the kidney.  The virus then continues to travel up the preganglionics into the spinal and where it goes transynaptic in the intermediolateral cell column (IML) to infect the sympathetic premotor neurons that are coming from the RVLM (and other brain regions).  The interesting part about using viruses is that the longer you wait following the injection (i.e. the longer the animal's recovery time) the further back the virus traces.  There is a limit, however, as the virus will eventually make the animals sick--it's a virus right? But by comparing shorter recovery times (i.e. shorter transport times) to longer recovery times (longer transport times), it's possible to see infection 1st in the post-ganglionics, then the pre-ganglionics, and then into the CNS.  Viruses have actually been used for a number of years to trace the sympathetic pathways from various organs (heart, kidney, adrenal, skeletal muscle, etc.; see Loewy AD and colleagues).  Another limitation of viruses, however, is that the interconnnectivity and complexity of most pathways can make it difficult to truly define specific connections of a subpopulation of neurons.  Until now.....

     The truly innovative aspects of this work is the use of a peudorabies virus (PRV) that contains a specific sequence (or cassette) that causes the cells to express a red fluorescent tag.  When the virus encounters a cell that also contains Cre recombinase (Cre) the red reporter gene gets taken out and the cell will now will express a yellow or light blue tag.  The sequence (or cassette) that causes the cells to express different colors is appropriately named the Brainbow cassette (yes like rainbow, see pic below).  What these authors were able to do was get the Cre recombinase to be expressed specifically in catecholaminergic neurons (yes, our favorite C1 cells).  So by injecting the PRV into the kidney, and waiting long enough, they could label neurons (in red) that went back up to the RVLM.  When the PRV infected a C1 cell in the RVLM the color changed to yellow or light blue.  This unique approach allowed them to label specific neurons in the RVLM that projected to the kidney (red) and a subpopulation of those cells that were the C1 cells (yellow or blue).
     This paper contains some really cool diagrams and pictures of the labeling so please check it out if interested in seeing it for yourself.  I would have posted them but I believe it would violate copyright laws despite the fact that the article is available for free online.  Finally, related to the lab, we have some high hopes for a compound known as wheat germ agglutin (WGA), which our colleague Dr. Goshgarian in Anatomy has used for transynaptic labeling instead of a virus.  We recently discoved that a company makes the WGA conjugated directly to an Alexa 488 fluorophore so there is no need to perform immunohistochemistry to see the WGA and make the process much simpler.  It's like combining the Fluorogold with a virus, hopefully the best of  both worlds.  Cool huh?