Hello.In this presentation we would like to show you how to do Pro Bois extension response assay in Esophagal. Probus extension response or PER is a taste behavior assay that has been used in flies as well as in honeybees. When the probus makes contact with an attractive substance, the fly extends its probos to consume the substance.

Solutions of various sugars such as sucrose, glucose, fructose, are very attractive to the fly. PR can also be observed by a contact made to the leg, but we would like to focus on the contact of the probos in this presentation. So the first step for PR assay is to starve the fly.

Optimizing the starvation condition is one of the most important factor for this assay. And there are two major things which are important here. One is the starvation time and the other is the body size of the fly prior to starvation.

And there seems to be a positive correlation between these two. And if the fly is big and fat, you need to starve the fly more. Now the body size depends on the nutrition of the fly food and the density of the flies in the vial.

So let me show you a good example. And you can see in this vial that the flies are not overcrowded. There are about, let's see, about a hundred to 150 PPE on the vial.

So as soon as the fly it closes from here, you would like to keep them in on a, in a fresh food for another three to five days, then transfer them to an empty vial, which has a piece of paper. This is kept moist all the time, and the starvation time can vary from 24 to 96 hours. We'll come back and describe about the starvation time later on.

Here's how to fix a fly for PER. First, you take one fly and an aspi aspirator. Then you place a 200 microliter yellow tip over the opening.

Now simultaneously flipping and blowing onto it. You can squeeze the fly up into the yellow tip gently apply some air to push the fly up to a point where it cannot move By using a razor, put a mark approximately at the fly's eye, then suck the fly back and cut off the tip. Now gently blow back, blow the fly back to position.

But just before reaching the very end, make a SL cut at the position where the probos is going to be located. You can do without the SL cut, but it would definitely make the following process easier if you had it. Now, cut the yellow tip tip below the fly and stuff the bottom with clay and place it upright.

You can make the final adjustment by adding more clay to the tip. One important thing is to try not to push the fly too hard. Let me just focus here and maybe I'll just push it a little bit further up.

So the fly's head would be a little bit above There. Now this is how it would look like under the microscope when you're trying, and this is a view from the side and the view from the front. And now you're ready to test pr.

The WIC we use to deliver the taste and is made in the following way. We use Kim wipes and we pass this through a shredder, which makes a strip like this. The width of the, the strip is approximately six millimeters, six millimeters, around five to six millimeters.

So now what you do is you twist this strip to make a thread, twist it tightly. So I'm twisting, and now we have a thread and so it becomes a thread like this. Next we take a pair of tweezers, pull, pull these small wigs apart from the thread.

And This is how we make wake. In this video clip, we would like to demonstrate the proper position to make contact to the Pro Bois. We will use a thread soak with 4%sucrose and we would like to show the difference in sensitivity by touching different regions in the dorsal ventral axis.

The center of the legum here is the, here is the most sensitive position. So you should always try to make contact with the to the center like this. Now when you touch the very top or the dorsal area of the labum, the fly wouldn't respond.

So we're making contact there, but you can see the fly is not extending the ssis. And the same thing happens when you touch the very ventral part of the labum. This is a little bit more difficult, but let's see if we can make a nice contact to the very lower part of the level.

We'll make a gentle, gentle approach. And let's see there. I'll touch the center again to make sure this fly is still in.

Interested in the sweet tasting. So in the lum, we should always touch the center, this region in the center. So when you look at it vertically in the assay, the center of the lum is pointing in this direction a little bit downwards.

So when you approach the legum just horizontally, which is in this direction, this direction, the closest point would be in between the sensitive and nonsensitive region. So that one respond. But if it's just just a little bit above, it's not going to respond.

So let me probe around. So now no response at the first row of silla, second row, third row, yes. So you have to go at least below the third row of the silla.

Now let me do the similar probing from the bottom. This is a little bit more difficult. I'm touching the second row from the bottom.

Now I'll move up there. The third row there, fourth row, no, it's not responding. Fifth row there.

So you want to touch at least the row number four to five Sula from the bottom. This all Means that you really want to touch the center constantly or otherwise it would greatly increase the variance of your data. You might want to practice this over and over again.

And the key factor is to make contact slightly below the closest point from a horizontal approach, which is here. Next I would like to show you the sensitivity difference between medial and lateral area of the labum. I move the position of the fly so that it would face up upwards in this picture.

First I will touch the center or the medial part of the fly slab beum, and you can see that the fly response nicely. Now, next I would touch the sensor located at the very lateral part of the labum there and you can see that the fly doesn't respond here there. Now let me probe from the side to the center and I'm touching the very side.

I'm moving toward the center little by little. And as you can see that as soon as it hits the very center, the fly would respond. Let me do it again from the side and toward the center there.

So it is important to touch the center of the level in both directions vertically and horizontally. In this section, I would like to demonstrate how to make contact with the osis. Generally speaking, you would like to make the contact as gentle as possible.

A good indication of the impact is the resulting head movement of the fly. So the ideal touch is something which you don't see the head being moved back at all. The other important factor is the shape of the wick.

I'll show some examples. The shape of the wick here is very good. It is not very thick, not very thin.

If it is too thick, it becomes hard to make a precise contact and the impact tend to be larger. If it is too thin, you wouldn't know if the solution is de delivered properly to the very tip, which is worse than a thick one. Let me touch the labum a couple of times with this.

And there, there, there, and you almost don't see any head movement at all by the contact there. Sometimes you get a wick that looks like this. So the tip part is clearly too thin.

You're not, you cannot be sure if the concentration on the tip is what you want to deliver. So what you can do with these is that you can use the yellow tip part and fold it couple of times like this is, and by doing so, you can make it into this nice shape with, let me make contact with this. So you can see that, that you don't see much head movements.

There was a little bit hip movement there, but yeah, this is another good example. You don't always have to throw away the thick ones, as long as you can make a precise gentle contact. You can still use these as well.

The head movement might be slightly larger than the thin ones, but as long as you have a steady hand, you can still make a a nice contact like this one. This is something you shouldn't use. There's, there are too much fiber coming out and it's hard to bundle them together.

You should throw away these and find another one. This one is also not suitable. The tip is too thin and too short to fold down to make a thicker one.

And the surrounding parts are too thick to make a precise contact. You should throw these away too. The Problem of this one is that it has too much liquid on it and it is hard to make a precise contact.

It would leave a larger amount of liquid on the prop Bois so it can interfere with the following procedure. Even if you had a very good wick, it wouldn't be good if you didn't make a gentle contact. Here's a bad example.

You can see the fly's head being moved violently. A strong impact would deter the fly from responding to a substance and the fly can easily lose interest to any following tastings. Next, I would like to give some tips about how to reduce the impact.

First, stop the wick above the yellow tip just before it makes contact. In this way, you can observe the amplitude of your hand shaking at the moment. And by looking at this, you can visualize the distance of the final approach, move the wick so that the far point of your handshaking would reach the illa of the vellum.

And of course it is better if you didn't have any handshaking at all. But this method allows you to work with it and it would reduce the overall impact. One thing we notice is that even if you start the fly for the same amount of time, you get a badge of flies that don't respond to anything at all due to insufficient starvation time.

Or on the other hand, you might find a batch of flies that are all dead. So testing flies from a fixed starvation time, regardless of the flies condition is not very wise. And it would increase the variation of the data so much that it can overshadow the phenomenon that are you, that you are looking at.

To improve this situation, we would like to introduce you to a series of controls to evaluate the state of the fly. The first step is to test the PER of a positive control, which is 4%sucrose. This concentration is high enough to induce a response.

For most star flies, if the fly does not respond to this positive control, that means either the fly needs more starvation time or it is inactive due to various other reasons like injuries. So you have to discard these flies. Now, if they did respond, try a negative control, which is water.

If the fly did respond to water, that means that they are thirsty or they just respond to everything or they're conditioned to eat anything that approaches. This is due to the attractive substance we gave as positive control here. And the fly learned that an approaching object is probably sweet.

So we are trying to measure a taste behavior, not a learning behavior. And you don't want the previous trial to influence the following trial due to learning. And it seems when a higher concentration is used as a positive control, the chances of a response to a negative becomes higher.

Now, you can test the fly for PER with whatever substance or condition you have, but after the test, you would want to try the positive and negative control again, because if the flight doesn't respond to positive here, that means that the it, it lost interest to stimulants during the test and you would not know if the non-responsive the test is due to lack of general interest of the fly. And next, if the fly responds to negative control, that means that the fly became thirsty or it got conditioned to an approaching object. And if the fly didn't respond to negative, you finally, you now have a data that you can accumulate.

So this window usually lasts about say five to 10, 15 minutes. And in some cases I've seen that this window can last up to 30 minutes. So after a while we realized that, so this is a graphic of relation between starfish time and mortality.

So this is time and this is the percentage of flies alive in a vial. And of course the they're gonna start dying and it differs a lot. And what, what, one, one thing I realized is that so till some certain point you there's, there's gonna be no flies that would respond to positive control, but suddenly you would start to see a fly that would start responding.

And of course that when time goes on, you can keep on using it. But from some certain point what we, what we realized that there's gonna be more flies responding to negative control. So this is gonna be the time frame that you want to catch in order to do ERs.

So the question is, when is this gonna happen? And in my experience, in a very good condition, usually this is gonna happen around 36 to maybe 48 hours, but sometimes this can go up to 72 hours and it really varies a a lot. So you really want to have multiple vials and set up so that you can catch this small window.

Once again, even with the same starvation times star, the same food, same everything, this really varies a lot. So even with the same genetic in a, in a very similar genetic background, this, this thing varies a lot. So that's the, the thing that you really want to be careful of.

We talked about the flies that were conditioned to the stimuli, which now responds to anything. And the one way to prevent this is not to give an attractive taste repetitively and to mix in negative controls in between. So let me show you how it works.

So this is a positive control. So that's 4%sucrose, we gave it twice here. And so if we keep this, keep giving a sweet sub this over and over again, it would eventually get conditioned.

So now instead of, so we'll mix in a negative control. So you see it, it's not re it's not responding to negative control. Let me do this again.

First positive control, 4%with 4%sucrose. Let me bend this a little bit. A very strong, very quick rest response once again.

And now once again, we'll go to negative control. And let's see, there's not a good, let me flip this around. Okay here.

So this is negative control water. And so you see it wouldn't respond to a negative control. Now let me show you a bad example.

So this is, we're giving 8%sucrose, so that's higher than the positive control that we use. So the fly really likes it and tries to seeks out, let me let, let us try again. Let's see a gentle contact there.

And it's very actively seeking out. It's still as its pros extendeds. So let's wait a while and let's make another contact there.

Oh, there. So it's, yeah, still looking for it. Looking for it.

Make sure, yeah, it's still okay. And maybe once more, once more, once again. Oh, there, yeah, it's very strong response.

Now instead of the 8%sucrose, let's give a negative control then. So let, let's bring this wick here. This is a little bit too thin.

So let me fold this and give to give, make a nice stair. But this is water. So you can see that in, you can see in this case where the fly responds to water.

Now there in this example, we gave a very attractive substance for five times. Now it doesn't mean that twice is okay, but five is bad. There are a lot of cases where the fly gets conditioned just by one application of 4%s gross or even a lower cost, lower concentration.

And the important message is to try to reduce the number of repetitive application of an attractive substance. And if you have a series of concentration of an attractive tasting, it is safer to start from the lower concentration and move up to a higher one, pulling back the taste and as quickly as possible so that the fly cannot consume much of it would also reduce the conditioning of the fly. Hello, in this presentation we've gone through all the steps for pro extension response assay.

The method itself sounds pretty straightforward, but just like many other behavior assays, there are small things that can prevent this assay to work. And we've gone through many of those steps that needs careful attention. Here I would like to reemphasize some of the most important factors.

And the first thing is the shape of the wick. And I've shown some of the pictures of very good examples, but in reality, everything is gonna be of this continuous spectrum of very good and very bad. And in the beginning you might be using something in the, this very good range, but by day, by day it might start shifting you.

The thing, the weeks that you might be using might start shifting to the not so good range. And the human perception of so-called good can shift very easily without, without noticing. And one way to prevent this is to take one good picture of a WIC that works very well and just look at the picture just before testing P.Now the second important factor is the impact.

And this is easier to deal with because the best contact, the impact, the best contact would be a contact where you don't see the head move at all. So it, if you can make it as gentle as possible, and if you don't see the flies head like shift in any ways, that would be the best. Now the starvation condition, the third thing, the starvation condition is the toughest factor to control.

And in many taste behavioral assays or olfactory behavior assays, a starvation time of 24 hours is used very often and 24 hours might be very convenient for a human for, for for us to do the experiment. But if you rear your flies in a very good condition, this 24 hours is rather short. But even if you use a very rich fly food and if you control the fly's density precisely, this window of experiment can vary from say 36 hours to 72 hours.Now.

So as an experiment, I have to say that it is very inconvenient for a human or people to do this experiment, but, and there's another factor that makes it tougher for experimenters. As a matter of fact, there would be many batches where the experiment window might be very short or even might not exist at all. So there are some major hurdles you have to go through.

But the most important thing is the positive control and the negative control. And by keeping those standards very strictly, you would be able to collect reliable data sets.