Midgastrulaステージショウジョウバエの胚からの神経培養の準備

My name is Diana Dowd, and I'm a professor in the department of Developmental and Cell Biology and anatomy and neurobiology at uc Irvine. And today I'm going to demonstrate the preparation of primary neuronal cultures from drosophila embryos. And what this entails is actually removing all of the cells from a mid gastro stage, drosophila embryo, and placing these in cell culture.

And what we use these cultures for are to really understand what are the genes and environmental factors that are important for regulation of electrical excitability and synaptic transmission between drosophila neurons. To start this procedure, we have to collect drosophila embryos. To do that, we take egg collection plates, spread a little bit of yeast paste on them.

It's a favorable substrate for the adult flies to lay their eggs. We put these plates on bottles containing a population of adult flies, usually a hundred to 200 flies. And then we let the flies, adult flies lay their eggs for about two to three hours.

Then we remove the egg collection plates, and these are the embryos that we use for the Culture procedure. The next step in the procedure is to Dec coate the embryos, and that means we actually remove the corion. We do this by washing the embryos off into a Buechner funnel on which we have a piece of filter paper so it catches the embryos.

We allow them to sit in the Buchner funnel in a 50%bleach solution. This 50%bleach solution not only dissolves the corion, but it also serves to, as a part of our sterilization procedure. We're doing this procedure actually not in the hood, it's outside the hood, so the embryos are, once they're dechlorinated, we wash them into a Petri dish.

They actually, when the Corian is gone, the viel membrane is quite sticky and it will stick nicely to a Petri dish. You can then pour distilled water on top of them and remove the distilled water two or three times and actually just throw the water off and the embryos will stick to the Petri dish. Don't use a tissue culture plastic Dish.

They do not stick to that. We begin the sterile part of the procedure by taking the Petri Dishes of embryos and taking it into a laminar flow hood. These are bellco cover slips.

They're uncoated, but they have been autoclave and they have not been washed. We find that they don't work very well. When they're washed, we take out four cover slips usually and put them in a single Petri dish.

So we will be making four embryo cultures in each Petri dish. Okay, so that Petri dish is basically ready for us to start culturing, and I usually make 12 cultures, 12 to 16 cultures at a time.Okay? The culture media that we use is a relatively simple defined medium.

It's A-D-M-E-M base, and we make this liquid medium up once every two weeks so it stays good for two weeks in the refrigerator. This has been sterilized. It's been put through a 0.2 micron sterile filter, and then we add five supplements to the media.

They are prepared once every two months and then frozen away in aliquots that are added to 10 mils of the liquid medium. So we just remove the contents. Final one, then this is just, we just gently invert this media to mix it up and we're ready to go.Okay.

Everything is set up. I have my culture dish that is ready, that or my Petri dish that has four cover slips in it ready to go. And now I'm gonna take my dish with embryos and I'm going to, they're now sitting in the distilled water.

I'm gonna remove the distilled water just by shaking it off. This is how I do it right into the trash can. And now I'm going to pour about two mills of the media onto these embryos, and now they're ready for me to actually insert the glass needle into individual embryos and remove the contents.

You have to have the media on the outside. Obviously if there was water out there, then you'd have osmotic shock. Now to get ready to actually prepare the cultures right before I remove the contents of the embryo, I actually put the five microliter drop onto the cover slips.

If you leave it too long, then the pH of the media changes. Okay, now I'm going to put a five microliter drop on the center of each of the four cover slips. It's actually important that these drops say stay as intact as they can.

If the media spreads all over the cover slip, then the cells are plated into some, a very thin layer of fluid is difficult. You want em to plate the cells into a nice round drop of fluid. As you can see there, we have four nice round drops of fluid ready to go.

And now I will take one of the pipettes I pulled and I then just attach it to this mouth pipette tube. You can use any tube you want, but the good thing about this tube, it actually comes with VWR pipe calibrated 100 microliter pipettes. And in these we have, there's a little rubber gasket here.

I can insert the mouth pipetter, I mean the glass pipette into this end of the pipetter. And then when I suck up the contents of the embryo, I'm not gonna be going up any further than this, the region where it starts to narrow. So there's this huge area separating me using mouse suction up here and the place where the cells are.

So there isn't, there's no problem with contamination. If you accidentally suck the contents up into this area, then you're going to have Huge problems of contamination. In our laminar flow hoods, we have Dissecting microscopes that are on scopic basis.

This allows light to be come in from below the embryo, and this allows us to actually see the cephalic furrow and the midgut and imaginations that you'll see when we look at the actual embryos. And this is how we actually pick out the stage of the embryo that is important for culturing to remove the contents of the embryo. We use glass pipettes, pull on a regular micro electrode puller that we use to make our patch recording pipettes.

We don't really care what the settings are. We pull quite fine pipettes because we're gonna break the pipette off in the dish next to the embryo at the size that we really want. We then take these glass pipettes, insert them through the viel membrane of the embryo, and using a mouth suction tube that's attached to the back of the back of the pipette.

We then extract the contents of the entire embryo. We then move it take, take the pipette out and move into another dish that has a cover slip with a five microliter drop of media. And we expel the contents of the embryo into that five microliter drop.

We pipetted up and down several times to disperse the cells, and then we let that cover slip sit in the dish for about 10 minutes Before we flood the dish with two mills of media. The dishes of cells after they've been Plated are put in a 5%CO2 incubator. This is very important because the media that we use is a bicarbonate buffered media.

We have some HEPs in there, but it's not enough to buffer at in the air environment. And so you have to use a 5%CO2 incubator. However, it has to be at a relatively low temperature around 22 to 23 degrees is ideal.

So we take a standard CO2 incubator that would be used from mammalian cultures and heat it to 37 degrees. What we do is we turn off the heater, we have it sitting in our lab, and we can put ice in the bottom of the incubator, and that keeps the temperature around 21 to 25 degrees. The other technique that we use is we take, again, one of these standard heating CO2 incubators and we put the incubator in a cold room.

Then you can actually heat quite effectively the incubator to 23 degrees if the ambient temperature is cold room temperature. And so those are the two methods we use to have a standard seal, mammalian seal to incubator to Allow ourselves to survive. All right, this is a Culture that was plated one hour ago at the same magnification that we saw right after plating one hour of after plating.

Here we see neurons that have differentiated for about two days. In culture, the neuroblast have divided one or more times and then given rise to neurons, which extend processes that form extensive overlapping neurotic networks works. Here we have two cell bodies that are head to head.

The top one is extending a process off at 11 o'clock, and the bottom is extending a process off at five o'clock. Those are their major extensions. And then you can see that they form finer branches.

These are very closely adhering to the glass substrate, and there are processes that you can see that come from other cells as well that are overlapping these. And these are sites of potential synaptic connection. We would patch one of these cells, and in general the, with this level of process elaboration, they would have synaptic functional synaptic currents.

Now our cells grow in culture 12 hours after plating. They will have extended pretty nice processes. Our standard physiology, we begin doing about one to two after culturing.

And you can see that they actually continue to grow processes for the next four or five days. And we have recorded from cells up to two weeks in culture, but most of our recordings are between Two and six days in culture with these cultures then that we've just Prepared from mid gastro stage drosophila embryos, we have networks of neurons that communicate in culture. We are able to actually look at the firing properties of these cells.

They have diverse firing properties. They have, they fire repetitive, some cells fire repetitively, other cells fire in birth, and these cells are primarily cholinergic or GABAergic. And we look at, we can look at synaptic currents that are mediated by these, by nicotinic, Aach, H receptors, and GABA receptors.