My name is, I'm A assistant specialist in Department of Anatomy and Neurobiology in the uc R one. And today I will explain you the procedure, how to do the wholesale recording on the PHA brain, the key procedure of this preparation as how to de captive head, how to remove the brain and how to remove the TRICARE system and The G clear tissue from the surface of brain. Today I will introduce you how to make A dissection solution for the whole cell recording a adult soft lab brain.
The first step is to make the, the second solution is really important, the key to make a good preparation for the adult brain. First I will put five mill of recorded solution in this cent field tube. And then here I have another small tube.
I will add one mill of recorded solution in this tube there, have some sing and then I will make this sing solution. I will pipe it up and down to make a good sing solution. And then I will take 100 unit of of pepane from this bottle.
And then I will put this pepane into this Centro field tube. And then you can see here the solution is cloudy and then I will put another 100 macro liter of cy and then I just mix that thoroughly. 10 minutes later the solution will be clear and all the crystal will be gone.
So this solution will be ready for dissection. So the best way to use this solution is 30 minutes after mix the cy And pepane. I will show you how to collect a Female fly from this tube because we have hundreds fly here.
But I will use this aspiration tube to catch the male and the female from here. But actually in the experiment we only use female in our recording. We prefer To use female fly to do the whole brain recording.
The reason female fly as bigger abdomen and white tape and the male fly has smaller abdomen and have clasps and duct tape. After I crack the female fly, I will decapitate the fly and remove the brain. Uses 2 27 gauge needles.
After I got the female fly, I will Use one needles insert into the CX ganglia and then fly will be paralyzed by this motor system. And then I will use one needles de captive the fly. Then I will put the head in the center of Petri dish.
Now I need to add one job of dissecting solution in each hand. I holding a one mill syringe with 27 gauge needles. Now you can see the pro side is on me and the dorsal side is on Norths.
This step I will make a slit by right needles to the right eye. Now I cut the ssis with the right needle and make another SL in the left eye that goes from the ventral to the dorsal surface. Now the brain have three slit and then I turn the head upside down with antenna sitting on the Petri dish.
And then I inserting the left needles to the space between the cuticle and the brain and gently remove the brain with right needles. The brain should come out intact with both optic Gloves and the pigmented eye tissue. Now I have decapitated the head and Removed the brain.
Now it's a good time to treat the brain with enzyme solution, contain pep pain and remove the TRICARE system. Now I want put the brain in the dissection solution, contain pepane for five minutes and you can see the dissect solution has been clear after sitting on the table for 30 minutes after intubating the brain and the dissection solution and the TRICARE system and GL tissue will be softened. So it's easier for me to use two number five very sharp forcep to take off all the trike and clear tissues.
It's important because if you cannot clear the triche and clear tissue, this tissue will clock your electrode. When you are approaching the neuron you want record, I will transfer the brain from this job of Dissection solution to this recording chamber in order to secure this tiny Brain. And the special design holder was used in this recording system to stabilize the brain on the bottom of the chamber.
So this special holder is made by a pum wear and then also have at least seven or eight nail cross hair on the pum wear. And the brain will be a pan killer to the cross hair and the cross hair will contact the region between central complex and optical locks. I will use force ship to put this holder on the brain and then by this method I can stabilize the brain on the bottom of the recording chamber.
Now I have secured a tiny pho brain in this chamber. And then now I will use this patch cla set up to do the recording on the neuron of this adult fly brain. And then I have a chamber perfused by 95%oxygen, 5%carbon dioxide and the solution will profuse through the chamber continuously.
I also have the two manipulator, two suter manipulator, and then I can do the dual cell recordings. Another I have fluorescence unit here and then all the GFP labeled fly neuron will be C under max cop and I can guide my electrode directly, go to that GLP positive cells and do the recording. I will show you how the brain secure in this chamber and how we record it that neuron from this preparation.
So this arrangement is really important. So under Max Mexico you can see the brain is holded by two cross hair and just contact the region between central complex and optical up. And then the brain is perpendicular to the cross hair and then it also incubated in this solution.
Now I want use this G for 1 4 6 US GFP flying to show you how I recording specific neuron in my patch CLA set up. You can see here that this is GFP positive neuron and max cop and all the project neuron labeled by GFP. And then I can use my electrode directly, go to this GFP plus neuron to patch on the cell and break in, make a wholesale Recording on this specific neuron because we use a genetic modified Fly to do our recording in this patch climb setup, we use a and U-A-S-G-F-P system to label all the all kind neuron that is specified labeled by GFP.
And we have done recording in the project neuron lock neuron and circadian neuron DPM neuron, all labeled by GFP by this can recording. We have find in this neuron there have very active synaptic transmission. Also have a very synchronized activity because we have done para recording on this group neuron.
So the next step is how to learn the mechanism under this kind of neuro transmission or or synchrony between two cell. We also want to correlate it this kind of neuro circuit activity to the behavior of the animals. And we also hope we can learn the basic mechanism from the fly and then tell us something, for example, how the Mammal brain system works.
