Forum - Basic Surgery FAQ

Bocarsly et al. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646561/) show using IBA staining that the inflammatory responses abates by 4 weeks, but not 2.

What time do you typically wait before commencing an experiment post-implantation?

Thanks for bringing this paper to our attention! It fits perfectly with our experience with our miniscope. If you try to image 1-2 weeks after implantation you will see absolutely nothing, and this is almost certainly because of the inflammation caused by the implantation. This image from the paper you referenced shows it beautifully (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646561/figure/g003/). Therefore we always wait at least 3 weeks before attaching our baseplate, and then it takes at least 1-2 weeks to habituate the animals to the weight of the miniscope, so our experiments generally begin ~5 weeks after implantation.

We will update our surgical guide with more information about this in the coming days, as well as add a section on baseplating.

Other question: what is the range of weights of animals that you have mounted the microscopes on?

We usually use male Taconic B6 mice around 12 weeks old, so they are generally 25-35g. We haven't had any mice that were too small to wear the scopes.

What microinjector do you use for viral surgery?

We use a nanoject II or III with capillaries pulled to a fine point. Our experience was that this decreases glial scar compared with something as invasive a Hamilton syringe, leaving the injection site still image-able.

We also use a nanoject with a Micro4 injector from WPI. Works great and we get very little damage at the injection site.

Hi, below a list of questions regarding the procedures:

a. For the virus injections, how many uL do you usually inject and what is a common titer of your viruses? Do you dilute them at all? According to the recent protocol by Stuber lab, they almost always dilute them (at least 1:1), but in other papers I don't see any mention of dilution. Do you do any long-term (i.e. 1-2 months after injection) recordings (in which case, according to the above protocol the high concentration can be problematic)?

b. When implanting the GRIN lens, do you lower it until it touches the surface of the hippocampus or is it fine if there is air/saline in between? I see in the wiki that you lower it 1.35mm, but I suppose this is dependent on the exact coordinates of the craniotomy, so I am trying to identify the general strategy.

c. According to the Stuber lab protocol, they cover everything with Black Cement. Do you also do this step?

d. In the wiki the steps for implanting the baseplate have not been uploaded yet, but is the general idea similar to the way described in the Stuber lab protocol (Fig. 14)? If I understand correctly, the procedure is done in a separate surgery because it needs to be done with the miniscope attached while observing the cells/FoV?

e. Are you planning to upload in the near future any videos of the surgery procedure?

Hi Nikolas, thanks for posting your questions!

a. For virus injections it is important to make sure your cells are healthy at the time of imaging (typically 1-3 months after the injection). As the nature protocols paper suggests, you should really check that the GCamp is excluded from the nucleus at these time points, because this is a sign of toxicity that should be avoided. This can generally be done by perfusing the animal and using confocal imaging. Unfortunately it's not possible to determine this from using the miniscope imaging alone. In our studies in CA1, we have not found the need to dilute the virus (AAV1-Syn-GCamp6f from Penn Vector), but this is very dependent on brain region and virus so I would recommend doing serial dilutions before you start the experiment to determine the best dilution for your experiments.

b. When lowering the GRIN lens, there should be a small amount of cortex buffer in the craniotomy to keep it wet so there should be no air beneath the GRIN lens. As you mentioned the depth of 1.35 is very dependent on your reference point. We use the most posterior point to reference. So we align the bottom of the grin lens with the top of the skull in the back of the craniotomy. This requires a bit of experience to get this consistent, and also a very good surgical microsocpe so that you can visualize it.

c. We seal the skull with Krazy glue, and then cover it with dental cement. We find the clear dental cement to be a better adhesive, so we use that, but black dental cement would work great as well.

d. Yes, exactly. There is a bit about it in our Online Workshop powerpoint presentation. I will upload a detailed protocol soon as well.

e. They are all available in the Online Workshop. Please let me know if anything is unclear. We will also planning to do a detailed voiceover for these ppts so that you can get all the tiny details that we can't put on the slides.

Hi Nikolas,

I just updated the Surgery and Baseplating Presentation link so go try again.