Just to add to the information mentioned above about WGA:

- It apparently tends to induce inflammatory responses in the injection site moreso than other tracer chemicals.

- It is allegedly taken up by some glial cells between first and second order neurons.

- Not only does circuit length (distance the tracer must travel) make things difficult in estimating survival time, but WGA transcytoses on a rather fast but variable time scale, ranging from one to four days. This effect seems to be concentration-dependent. You'd have to test out time courses and concentrations in several mice to determine the optimal transsynaptic spread. 

With its bidirectional spread, I'm not sure that WGA would be appropriate. You wouldn't be able to isolate transsynaptic anterograde labeling from transsynaptic retrograde labeling. You may want to look into using recombinant Tetanus Toxin C Fragment (TTC), which is preferrentially retrograde and transsynaptic, and tracing from PFC back to vHipp. The main issue there is that recombinant TTC is prohibitively expensive to purchase.

One last general thing - using IHC staining will provide superior visibility of labeled cells compared to visualizing the fluorescently-conjugated tracer as-is (in most cases). This may be important if transcytosis substantially dilutes your tracer over synapses.

I don't think the distance is a huge issue, since I have injected WGA into saphenous nerve (in the distal hindlimb of the mouse) and gotten good labelling in dorsal root ganglia (although it didn't have to cross a synapse to get there). But, WGA is definitely both anterograde and transsynaptic, so I think it's worth a shot.

Hi, anterograde, retrograde, and trans-synaptic properties are species-dependent , and definitely distance dependent if you are talking about great distances (i.e. mouse versus monkey).  not sure which species you are asking about.

it works in the mouse brain for most of the applications but tends to be quite weak and  only a fraction of second-order neurons are actually labelled

Great- thank you all for your input. I am using a mouse model. Francesco- would you have a suggestion for a tracer that may be a better option for identifying the second order neurons?

Just to add to the information mentioned above about WGA:

- It apparently tends to induce inflammatory responses in the injection site moreso than other tracer chemicals.

- It is allegedly taken up by some glial cells between first and second order neurons.

- Not only does circuit length (distance the tracer must travel) make things difficult in estimating survival time, but WGA transcytoses on a rather fast but variable time scale, ranging from one to four days. This effect seems to be concentration-dependent. You'd have to test out time courses and concentrations in several mice to determine the optimal transsynaptic spread. 

With its bidirectional spread, I'm not sure that WGA would be appropriate. You wouldn't be able to isolate transsynaptic anterograde labeling from transsynaptic retrograde labeling. You may want to look into using recombinant Tetanus Toxin C Fragment (TTC), which is preferrentially retrograde and transsynaptic, and tracing from PFC back to vHipp. The main issue there is that recombinant TTC is prohibitively expensive to purchase.

One last general thing - using IHC staining will provide superior visibility of labeled cells compared to visualizing the fluorescently-conjugated tracer as-is (in most cases). This may be important if transcytosis substantially dilutes your tracer over synapses.

Kevin - Thanks so much for your thoughtful answer. Because of the type of connection that I'm studying and the distance, I feel like pollution from retrograde labeling, injection site inflammation, and glial uptake will be minimal at the site I will be co-staining. I do need an anterograde tracer, which is unfortunate because I feel like there are perhaps more options for retrograde. 

Another consideration- if I will most likely need to do IHC with an anti-WGA or anti-Fluor anyways to improve visibility of the second order neurons, would I just want to inject an unconjugated WGA and therefore perhaps improve transsynaptic mobility? Then I would do the same IHC I was already planning on doing... 

You are right on the unlabeled WGA - it should have greater mobility when unconjugated. Go with that and then you can amplify the stain further (with labeled streptavidin method) if necessary. I've used a decent antibody for WGA from Vector labs at around a 1:10,000 dilution, though your mileage may vary.

unfortunately, options for anterograde second-order neurons labelling are quite limited....