Measuring Exocytosis in Neurons Using FM Labeling

I am Jimmy Lynn. I'm a graduate student in the Molecular and Cellular Biology program at Harvard. I'm currently working in Murphy Lab and we work with Hippocampal Neuro in the lab.

And right now I'm gonna be showing you how to do FM d experiments. And so now we're gonna go ahead and mount the cover slip that has neurons on it into this custom made chamber. Our microscope is an inverted CC, D and make sure to thoroughly dry off the bottom.

No going to connect the electrodes. No.Now the profusion is set up. I'm gonna place it at one end of the chamber.

We Use clay in order to hold. The leaders of HBS per experiment will go ahead and add all of that. Fusion is set up.

You wanna place use clay in order to hold this in place, I guess like this that has cell bodies because the membranes of the cell bodies will pick up a lot of the dye and be a lot right into the synapsis. Maybe an adjacent area like here where just this area I had the immediately and we're going. And so Initial photo bleaching from the first 10 images.

And then here's where, so I'm Jamil Newton. I'm a graduate student in the molecular biology program here at Harvard. I currently work in a neuroscience lab with Vinky Murphy as my pi.

And what I wanna talk to you today about is doing FM experiments. And what is your, what is the goal of your experiment? And so the nice thing about FM is FM is a fluorescent dye that allows us to track the kinetics of exocytosis.

And so we first make sure that the dye is taken up into the synapsis by giving it a loading stimulus. And then after a wash period, we give it a detain stimulus in which the dye is released from the vesicles. Are you working on specific type of neurons?

And so within our lab, we use this for hippocampal neurons and we use this in cultured neurons rather than in lysis. What are the possible problems with this experiment? And so some of the common problems, what we use is electrical simulation.

Sometimes in the past, for whatever reason, there's been problems with getting the electrical simulation to work. And I know that other labs also use high potassium solution. So high potassium solution can be a nice alternative to actually using the electrical stimulus if there's problems there.

Also, typically, for whatever reason during the wash step, it seems like you're not getting enough release of the nonspecific staining. And I've often found that just by increasing the flow per rate, the flow rate, and so rather than half an ml doing an ml and a half per minute, and by increasing the amount of time from like five minutes to a 10 minute watch dramatically improves the results. What are the possible applications of this experiment?

What do you actually want to study? Okay, so you can use it to test control cells versus cells that are somehow manipulated. And so for example, different lines of mice, you can see if the kinetics of release are somehow different or you can see if you add a certain drug if that actually affects cytosis.

Thank you.