I do LTP recording in rat hippocampus slices. I have been doing it for a while but off late I am facing a couple of strange issues.
1. The slice is thinning out with time while it is in the recording chamber. I use 400 um thick slices and do surface recovery for 2.5hrs before I transfer it to the recording chamber. While placing it in the chamber, the slice looks healthy. I then do a 20mins in-chamber recovery after placing the electrodes and then start the input-output to figure out where to clamp the current. Everything goes fine till this step. Once I start the baseline recording, the slope remains steady for 5-10 mins and then starts to fluctuate a lot. I have seen a trend where it increases with time. When I look into the microscope to check if the slice has moved or is in normal state, I see that the slice has almost become transparent because of thinning out. It's as of the upper layers of it have been scraped off. A couple of times it had become so thin, I literally had to scrap it off the floor of the recording chamber. I cannot figure out why it could be happening. No one in my lab had experienced this before. The first time it happened, I suspected there could some some detergent residue in the aCSF container. So I have been washing it several times with milliQ water before use. Yet the problem continues.
2. Several times, even at very low current, bubbles appear in the slice near stimulating electrode. I have observed this happening at higher current, but this is happening at very low current lately. Not just that, last two stimulating electrodes broke in a very weird manner. I am attaching the pictures of them. In past, electrode got damaged due to physical mishandling, but usually the tip gets bent or the two electrodes are not parallel to each other. But never have I seen an electrode break in this manner. It was more like it got corroded. The second electrode was made just 2 days ago and was used for just 2 experiments. Neither of them hit the floor or the walls of the chamber and yet got damaged.
I cannot figure out why this could be happening. No one I know has ever seen anything like this. If someone has experienced the same, I would be grateful to know how you could fix the issues. I would also be grateful if you can help understand the possible reasons for why this could happen.
Thank you in advance.
Hi Deepti,
This seems to be an issue with the stimulation electrode or the isolation box. At least, based on the 2nd point. Try to replace each one at a time to see where the problem is emerging. I think the current you are giving is much more than what it should be. Is this an interface or submerged chamber? In the interface chamber the stimulation intensity you need to give is much less than the submerged condition.
"bubbles appear in the slice near stimulating electrode"
I agree with Gürsel that you isolation stimulation box might be 'continuously leaking' current and/or not delivering a constant value. Best solution if your Lab. has money: get another one! Price around a $1000 (pure guess). Battery operated stim. box (E.g., Iso-flex), do need batteries, so check V-output. Should be equal or a bit more than 90 V. Maybe changing the batteries could do the trick. Could be an electrode problem, but I am guessing you have put a new one and still see the same problem.
Also based on your description of slice thinning. Maybe, just maybe...Do you have your 2.5 to 3 mM K out? a lack of K would not be good for the slices as they appear a little weird.
Dear Deepti
For your first question
I did not understand your "thinning out" concept clearly. In general, when you cut slices at a set thickness, during recovery as time goes on, there will be a difference in the thickness depending on the osmolarity changes. I would not worry too much about this. There are several parameters that cause fluctuations in the slope or amplitude values in an extracellular field recording. The most important factors are having a consistent flow of your perfusing aCSF, good amount of Carbogen bubbling and the room temperature. The age of the animal is also a critical factor in getting a stable recording. During my PhD, I worked with 4-5month old rats and I had to wait nearly 30-60mins until I see a stable baseline for 20mins. It's all about patience. Don's give up when you see a fluctuating baseline, in most cases it gets stabilized as long as the above-mentioned factors are consistent.
Also keep in mind that for the extracellular field recordings, the depth of the aCSF in the recording chamber should be kept low, I would say around 1-1.5mm.
For your second question
What kind of electrodes are these? Are you using platinum-iridium bipolar electrodes? These electrodes generate bubbles proportional to the increased current strength because that's how these electrodes work. Platinum catalyzes the electrolysis of water. The gaseous H2 or O2 produced, depending on the direction of current flow, leaves the surface of the electrode. Refer to Axon Guide, Fig 1-9, page number 22. You can download the Axon guide here https://www.moleculardevices.com/en/assets/user-guide/dd/cns/axon-guide-to-electrophysiology-and-biophysics-laboratory-techniques
Are bubbles generated continuously or only during stimulus?
Good luck!