Does anyone know of any intracellular solutions or methods for reducing or preventing LTP washout after going whole cell? Canoncial thinking suggests that after 5 minutes dyalisis of the cell resulting from the pippette solution starts to "washout" key molecular components of LTP. I would like to extend this time window if at all possible to give drugs in my pippette solution time to diffuse throughout as much of the cell as possible. Along with this, are there any studies that have measured the diffusion kinetics of drugs introduced into cells via patch pipettes? This would be in pyramidal neurons, area CA1. I'm sure people have done this with florescent labels etc. just not sure if the kinetics would be the same for a drug of interest.
Personally, I think the dogma of "5 minutes" is a bit over sold, as I've seen 50% LTP after 15-30 minutes of whole cell. That said, some people still get pretty worked up about this concept. What I would typically say is "patch with high resistance pipettes", but seeing as you want to diffuse in a drug, I think you should try another approach. Go in, patch the cell, hold it for a couple of minutes, then pull off. Wait for half an hour/45 minutes, then go in and patch it again. I know it's not perfect, but I think it's a good happy medium. Give it a try with some Alexa to see how it compares to just holding the cell for 45 minutes.
Hi Steve,
I agree with what William said above. Just to add you may want to try perforated patch clamp technique. I use a less invasive method in which I use simple voltage steps set on the amplifier to break the membrane instead of using suction which may suck too much out of the cytoplasm and enzymes-substrates required for normal cellular function. In this method, once I seal to the cellular membrane I apply voltage steps until I break the membrane with no suction at all. What I find is that the cellular responses and the washout of cytoplasmic content is a lot slower and the survival rates a lot higher. For details please have a look at my following publications.
Best wishes,
Refik
Article Semi-loose seal Neurobiotin electroporation for combined str...
Article Neurobiotin Electroporation for Combined Structural and Func...
Hi, I am not sure what pipette solution you are using at the moment, but I am wondering if you have some calcium in there? I know standard pipette solution usually do not include calcium, however, calcium can be important in terms of signaling cascade. I have found that with physiological calcium concentration (~ 100 nM), you can get pretty good patch recording without compromising patch quality.
https://www.ncbi.nlm.nih.gov/pubmed/20423724
- Badges
- Science topic
- Similar topics
- Biological Science
- Cell Biology