My name is Diana Dowd and I'm a professor in the departments of Anatomy and Neurobiology and developmental and cell biology here at uc Irvine. Today I'm going to tell you about the preparation of neuronal cultures from Drosophila Pupi. This involves removing the brain from late stage pupi and we generally culture just the central brain region.
And at this stage, the major anatomical structures that are present in the adult brain are already formed. This includes the antenna lobes where there are projection neurons that send their axons all the way up to the mushroom body region and the mushroom body neurons, Kenyan cells, which have sent their axons down to their target neurons through the Alpha, beta and gamma lobes. On the day of culturing, The first thing we do is go into the laminar flow hood and we prepare sterile, the enzyme solution that we're gonna use to dissociate the tissue.
And we also make up the medium that we're gonna use for growing the cells. And this is made from stalks that we've prepared ahead of time. The next step is to collect the pupi that we're gonna use for this dissociate or for the dissection.
And that's done outside of the hood because these are grown in vials that contain yeast and all sorts of other things. And so we actually do the initial stages of our dissection Outside of the hood. These are peepee that I've just taken off the sides of a vial, and on this left hand row we have animals that have red eyes and they are a good stage for culturing.
These ones are slightly, these PPP over here are slightly younger. They have orange or yellow eyes, and this is an okay 1 0 7 line. They have slightly brighter eyes than regular wild types where the color at these stages would be a little bit more brown.
All of these stages are fine for culturing, for making the people Brain cultures. Okay, the first step here is To put a couple of drops of dissecting solution right next to the pupi that I have pulled out and lined up in my Petri dish. We use the tops of 35 millimeter Petri dishes.
I then take a pair of no number five forceps in my left hand and a one CC tuberculin syringe with a 27 gauge needle at the end. These are my two dissecting tools. And to remove the head from the pupil case or from the pupi, basically I have to use the i, I hold the pupi with my forceps in my left hand, and then I use the needle in my right hand to remove the front of the pupil case.
I then put the pull back the the cuticle covering and the head pops out the front. I use my needle then to sever the I at the neck region and I pick up the head and place it into the drop of dissecting saline. I will repeat this procedure 10 to 15 times because to make for a good culture, I like to have 10 to 15 animals of each genotype.
All right, I'm going to Remove the brain from the head of the animal. To do that, I use two tuberculin syringes each holding a 27 gauge needle. The left needle I place right through the animal's psis to pin the head down to the Petri dish bottom.
And then I use my right needle to open a slit on the right side, and that is where I'm going to push the brain out. Okay, and I'm, the next step then is to put my left needle over the the left eye. And now I'm going to actually just ex push and the brain should come out the right side through the slit.
Here it comes. You can see it on the right hand side, and now I can just use my right needle to separate it from the rest of the head capsule and the eyes.Okay. Now, before doing the final dissection, which is to remove the optic lobes, I transfer the brain to a clean drop of saline.
I can either use a pipe eman to do that, and I pick it up in the very tip of a 20 microliter pipe eman, and I use it set at five microliters, or I can actually transfer it on the needles As well. This is the last stage in The dissection procedure. It's done in a fresh drop of saline, and generally I'd have about 10 to 15 brains in here.
I then remove the optic lobes from each one by using the 27 gauge needles as cutting tools, and I just sever the left optic lobe and then I sever the right optic lobe on each brain. And at this point, all of the brains that have been processed this way then will be transferred into an tube that contains the enzyme solution, which will be used for the enzymatic dissociation. All of the brains from a single genotype are put into a single epi screw cap einor tube that contains one mill of the enzyme solution.
And then we just stick them into a a test tube holder and put them on a rotator where they sit for 10 to 15 minutes at room temperature. And that's the actual time of Dissociation. To complete the Enzyme dissociation step, we actually have to remove the enzyme.
So we bring the tubes over to an EOR centrifuge, place the tubes in the centrifuge, and we are going to spin them down for three minutes at 3000 RPM. After we spin the brains down, we bring them now to our laminar flow hood. This is where the sterile part of the procedure begins.
We now don't open the tube unless we are in the laminar flow hood, and we will now remove the enzyme solution. We'll replace it with dissecting solution, and then we will spin and wash a number of times with both dissecting solution and ending up with a Defined medium, which is what we grow the cells in. All right.
The first thing I Need to do here is I need to put a five microliter drop of saline on each of the cover slips that I have in these Petri dishes. There's a single cover slip per Petri dish, and it's been coated with con a laminin, so I put a five microliter drop right in the middle of the cover slip. That's where my drop of laminin is.
It's very important cause you want to concentrate your cells right in the middle of the field because we get about 10, 000 cells per brain, and that is what we make a single culture with. Okay, so now looking in the microscope, here are nine brains that have already been gone through the enzyme dissociation procedure. You can see they're still pretty much intact.
They don't have their optic lobes. Those were removed, but we just have the central brain region and now I'm going to pipette up a single brain and move it over to the five microliter drop on a cover slip. And I do that four times.
I take two one CC syringes put on the 27 gauge needles. Again, it's really important to note that these are very cheap and disposable dissecting tools, so undergraduates can do this without worrying about your budget. Okay, so now I have the two needles and I'm going to do the initial mechanical dissociation, which is to just cut this brain up into several pieces, which will allow me to actually suck these pieces up into a glass pipette in just a minute to complete the final dissociation.
When I'm doing this, I don't get them down to tiny pieces, I just want to increase the decrease the total size so I can get a little bit so that the pipette, when I do the dissociation brings up slightly smaller pieces than the full brain. I also wanna do it relatively quickly because you see the drops of media are small. That means we have a high surface to volume ratio and the both the pH and the osmolarity of the solution can change if I leave it sitting out for any length of time.
When I was first doing this, I would only actually do two brains at a time, two cultures at a time. Now I usually do four, and now I'm gonna break the pipette, but I can't talk about it or anything, right? This one I've just finished.
The dissociation, the size of the pipette was pretty good. I was able to suck the tissue up and up and down a couple of times, and you can see individual cells and those, we still have clumps, but that's okay. They'll wash away.
Now, the cultures, as soon as they've been plated, are placed into a second Petri dish and they're placed in the CO2 incubator within a couple of minutes because we wanna make sure that we maintain the pH and the osmolarity at a level that supports their survival. So we stick em in here for 30 minutes before we flood the dish. These cultures were Prepared from the brains of late stage pupi.
The culture has been grown for two days in a CO2 incubator, and you can see individual cells that have extended processes. The extent of process elaboration is less than what you would see at five days and more than What you would see at one day. My name is Jorge Ano.
I work in the Doubts lab. I'm gonna show you right now how we do the experiment, the calcium imaging experiment in the people cultures that we just prepared. This is a video of a calcium imaging experiment where you see blue cells showing low, indicating low calcium levels and red cells indicating high calcium levels.
We'll see that there are some cells that turn to red and it indicates intra increasing intracellular calcium levels. And those are spontaneous and those are transient. They go back to to Baal calcium levels over time, and after a while, we're gonna apply a drug, in this case, nicotine, and you will see the cells increase the intracellular calcium levels.
We apply nicotine for 100 seconds, then we watch out nicotine, and then the cells go back to basal calcium levels. So the real advantage Of this culture system is that we're able to make our cultures from both transgenic and mutant lines of flies, different ones in which different subpopulations of neurons are labeled with GFP. This is the brain from one of the lines we've used in which GFP is expressed in the mushroom body, Kenyan cells, neurons involved in mediating complex behaviors, including learning and memory.
The cell bodies are located up in the dorsal aspect of the brain around the region where the red arrows are. And these L-shaped structures that are GFP positive are actually the axonal projections from these mushroom body Kenyan cells. The after dissociation of this brain tissue.
The neurons regenerate their processes in culture, and you can easily identify the GFP positive Kenyan cells in this case. These are accessible to both then standard electrophysiological recording and calcium imaging studies. Therefore, these cultures are really useful tools for exploring the genetic and environmental factors that are important in regulating function of neurons that we know are important in mediating complex behaviors in the Adult animal.
