Argh, today was an exceptionally frustrating day. 
For the last week and a half I've been planning on doing a big series of electrophysiological experiments - I made new recording dishes (which is a giant pain) new culture media and solutions, new extracellular and intracellular recording solutions, new microelectrodes and perfectly timed cell cultures so that I could begin my experiments today.
My idea is this: I've developed an in vitro recapitulation of the mammalian inferior olive -> deep cerebellar nuclear synapse and have been testing it for a while to see what sorts of neurotransmission happen there. I've made an interesting finding - if I briefly pulse a solution containing moderate concentrations of nicotine on the cells for about five minutes, all excitatory (but not inhibitory) neurotransmission in the dish gets faciltated - meaning there is more of it, and it's stronger when it happens. I want to figure out exactly how this occurs, so I'm going to sequentially block all of the nicotinic acetylcholine receptors using toxins (these are presumably what's speeding up neurotransmission) and then pulse the cells with nicotine and record. Presumably, when I've blocked the proper receptor(s) I won't get the same facilitation of transmission. Once I've identified which receptors are responsible, I want to look at cultures taken from our genetically modified mouse lines that lack a protein that is involved in regulating the localization and expression of nicotinic acetylcholine receptors and see if they have the same response. Pretty straightforward experiment, but it would be really cool if it worked.
So I got into the lab around noon, and spent about two and a half hours getting everything set up, including my new stimulating electrode and all of my perfusion solutions (which I also had to make.) Finally I got my cells and set them up for recording. And I couldn't patch any cells. I tried and tried and tried for almost six hours, and never got a single recording from any cell. It's so fucking annoying, I set everything up in advance, had all of the components ready to go, and now can't do the most basic part of the experiment.
I have two more days to try until the cells are too old and I have to start the whole experiment over again.
Sorry about the rant there, especially a rant that I guarantee no one can actually relate to, but I'm pretty pissed off at my wasted Sunday and I needed to vent.
For the last week and a half I've been planning on doing a big series of electrophysiological experiments - I made new recording dishes (which is a giant pain) new culture media and solutions, new extracellular and intracellular recording solutions, new microelectrodes and perfectly timed cell cultures so that I could begin my experiments today.
My idea is this: I've developed an in vitro recapitulation of the mammalian inferior olive -> deep cerebellar nuclear synapse and have been testing it for a while to see what sorts of neurotransmission happen there. I've made an interesting finding - if I briefly pulse a solution containing moderate concentrations of nicotine on the cells for about five minutes, all excitatory (but not inhibitory) neurotransmission in the dish gets faciltated - meaning there is more of it, and it's stronger when it happens. I want to figure out exactly how this occurs, so I'm going to sequentially block all of the nicotinic acetylcholine receptors using toxins (these are presumably what's speeding up neurotransmission) and then pulse the cells with nicotine and record. Presumably, when I've blocked the proper receptor(s) I won't get the same facilitation of transmission. Once I've identified which receptors are responsible, I want to look at cultures taken from our genetically modified mouse lines that lack a protein that is involved in regulating the localization and expression of nicotinic acetylcholine receptors and see if they have the same response. Pretty straightforward experiment, but it would be really cool if it worked.
So I got into the lab around noon, and spent about two and a half hours getting everything set up, including my new stimulating electrode and all of my perfusion solutions (which I also had to make.) Finally I got my cells and set them up for recording. And I couldn't patch any cells. I tried and tried and tried for almost six hours, and never got a single recording from any cell. It's so fucking annoying, I set everything up in advance, had all of the components ready to go, and now can't do the most basic part of the experiment.
I have two more days to try until the cells are too old and I have to start the whole experiment over again.
Sorry about the rant there, especially a rant that I guarantee no one can actually relate to, but I'm pretty pissed off at my wasted Sunday and I needed to vent.
VIEW 7 of 7 COMMENTS
you know what you need? some tough love. tell those cells if they don't patch, yr gonna... do something really mean!
So sometimes seeing big words like electrophysiological make my brain cramp.
over & out.
i plan to make some of these meetings public events for exactly that reason.