It sounds like the CAV-Cre + AAV-DIO-opsin virus combo would be best.

I've used WGA alone as a tracer in the brain and via virus as AAV-WGA-Cre, and it kind of goes everywhere. WGA is quickly transported transsynaptically retrogradely and anterogradely through most neural circuits. Thus, the issue that occurs is whether the DIO construct is being expressed in first, second, or even third order neurons in relation to the AAV-WGA-Cre injection site. For this reason I'd avoid AAV-WGA-Cre, regardless of serotype.

Replication-incompetent PRV would work better as it would go monosynaptically retrogradely (depending on serotype, I think), but it requires biosafety level 2 facilities and quarantine protocols. I also haven't heard of labs that distribute PRV-Cre viruses, though they may exist.

The CAV virus infects cells monosynaptically retrogradely, it only requires BSL1 facilities, and there is a distributor for it in France. I personally haven't used it but I know people at my university that have used it in combination with a Cre-dependent AAV construct (DIO-DREADD instead of opsin) with reasonable success. The timeline is the same as a non-Cre dependent AAV for optimal opsin expression, about 4-6 weeks post-injection.

Good luck!

I just recalled two additional issues with isolated WGA that would probably arise in cases where neurons produce it through AAV transfection: 

1. After escaping axon terminals, WGA also invades glial cells. 

http://www.ncbi.nlm.nih.gov/pubmed/3611421

2. I read somewhere that WGA injected into the brain (as a tracer) provokes an inflammatory response. I can't seem to find the reference for this, which I believe was a book from decades ago... 

Just thought I'd add this info, in case it helps.

In theory it could, but you'd need a promoter in the CAV's code that is used by that specific cell population. I don't think CAVs with specific promoters are available, though...

Hi Adam,

We have tried AAV-WGA-CRE (from UNC) in mouse, and while you see infection in the injected region (mCherry reporter expression), we never saw any retrograde expression. Speaking with others at conferences, no one I have spoken to is getting good expression with WGA-CRE.  We and others have had much more success with HSV or CAV for retrograde Cre expression. 

As for the AAV serotype, you really will need to validate it for your own particular brain area. Also, there has been some suggestions that certain AAV serotypes (AAV2.1 aka AAV6 & AAV5) are themselves retrograde, but again you would need to validate this first in your particular system.

Hi Adam,

we injected AAV-WGA-CRE into mouse retinas hoping to achieve transsynaptic anterograde transfection of thalamic neurons (similar to what has been standard in the field for a _long_ time using WGA-HRP).

No success at all.

I would also like to second what Eoin C O' Connor just said: Also from my discussions with many people on conferences etc. I get the feeling that transsynaptic transfection with WGA-Cre is capricious (to say the least). I did not hear much (actually: anything at all) about TTC-Cre, though.

We use RABV (glycoprotein free) for the retrograde and anterograde tracing. I do not know you can combine opsin with this virus and inject in the specific region!

I tried WGA-cre with DIO opsins in rats. The success depends on the particular pathway you are targeting and I would agree with the previous comments that this virus is quite capricious.

I saw nice expression in the BNST cells projecting to central amygdala, but not in the other way around. In cortex (insula, mPFC) to BNST pathways, I saw some labeld cortex cells: but not as many cells were infected as you would expect knowing the results of the traditional tracing studies...

You definitely need to check for retrograde jumps problems. It might not be possible to do whole brain cuts with every experimental animal, but doing one check with every new aliquot / surgery group is a good idea.

Also, I was told by the UNC Core technicians to use low protein binding tubes to store the aliquoted virus and never aliquot smaller than 5 uL for big virus like WGA-cre.

Hi! I am quite interested in using the WGA-Cre but as it sounds like it seems not as consistent as I expected it when using viruses. Is there a publication where WGA-Cre has been brought in the mouse as a transgene activated via Cre or Tet or other systems to label the network of the activated cells with a Cre Lox reporter?

Thank you very much!

Hi

I copy an excerpt from (Dautan et al 2014 J neuro). The wheat germ agglutinin (WGA)–Cre fusion protein was expressed by the AAV–EF1a–DIO–WGA–Cre–mCherry, transcellularly transported to the afferent neurons, and retrogradely transported to the soma, in which it produced the recombination of AAV2–EF1a–DIO–hChR2–YFP and the expression of YFP.

I am similarly interested in trans-cellular viruses but I would need one that moves anterograde. From what I gathered here viruses expressing the WGA might not be ideal.

I talk with the first author of the paper mentionned in my previous reply and he confirmed that the AAV–EF1a–DIO–WGA–Cre–mCherry was really AAV–EF1a-WGA–Cre–mCherry (not cre-dependent). I guess there is no cre-dependent WGA virus yet then.

Dear Researcher,

Utilizing Wheat Germ Agglutinin (WGA)-CRE virus for the specific activation of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) or optogenetic opsins in projecting regions of neurons is an innovative approach that leverages the anterograde trans-synaptic tracing ability of WGA. This technique allows for the targeted manipulation of neuronal activity in brain circuits, particularly useful in neuroscience research focused on understanding complex neural networks and behaviors.

When employing WGA-CRE virus to activate DIO (Double-Floxed Inverted Open reading frame) opsins in projecting regions, several critical considerations should be taken into account:

Viral Vector Selection: Ensure the WGA-CRE viral vector is compatible with your target species and cell type. The efficacy of viral transduction can vary significantly between different neuronal populations and animal models.

Titer and Volume: Optimizing the titer and volume of the viral injection is crucial for achieving robust expression in the desired projecting regions without causing toxicity or inflammation. Pilot studies may be necessary to determine the optimal conditions for your specific experimental setup.

Injection Site and Technique: Precise injection into the source region of the projections is essential for successful targeting. Utilize stereotaxic surgery with accurate coordinates to ensure the virus is delivered to the correct location. Additionally, controlling the injection speed and allowing time for the virus to diffuse can improve transduction efficiency.

Temporal Considerations: WGA-CRE mediated trans-synaptic transfer and opsin expression have a temporal component that must be considered in experimental design. There is typically a delay between viral injection and optimal opsin expression, which can range from several days to a few weeks depending on the system. Planning experiments should account for this timeline to ensure peak expression coincides with data collection.

Validation of Targeting: It's imperative to validate the specificity and efficiency of opsin expression in the projecting regions before proceeding with functional assays. This can be achieved through immunohistochemistry, fluorescent microscopy, or functional assays such as optogenetic stimulation or inhibition followed by behavioral observation or electrophysiological recording.

Controls: Implement appropriate controls to discern specific effects of opsin activation from nonspecific effects of the virus or the surgical procedure. Controls might include animals injected with a non-CRE expressing virus or animals lacking the DIO opsin but subjected to the same experimental conditions.

In summary, the use of WGA-CRE virus to activate DIO opsins in projecting regions is a powerful technique for dissecting neural circuitry and function. Successful application of this approach requires careful consideration of viral vectors, injection parameters, timing, and rigorous validation of targeting specificity. Sharing experiences and challenges within the research community can further refine these methods and enhance our understanding of complex neural phenomena.

Best regards in your research endeavors.

With this protocol list, we might find more ways to solve this problem.