Hello everyone,
I am currently performing calcium imaging experiments in rat acute brain slices. I have been doing this type of experiment for a while now and never had any problems with bath applications of drugs. However, I am now working with drugs that I cannot apply via bath because they are very expensive. Therefore I am trying to apply drugs with a patch pipette positioned near the cells of interest.
However, I am facing wo major issues :
- the pipette gets clogged very often (unlike with patch clamp, i cannot apply a positive pressure when going into the slice because it would apply the drug too soon and desensitize my receptors). Therefore I'm not always sure I do apply the drug. To be certain of whether the drug is applied onto the slice, I added sulforhodamine 101 to the pipette to monitor drug application, but it doesn't fix the clogging issue...
- I get a lot of purely mechanical responses to the application, even with very low pressure applied . Some cells display calcium transients almost immediately after the start of the application, whereas the response I'm looking for should take longer since it involves GPCR activation).
Does anyone have advice on how to overcome these issues ? Thank you in advance !
Ivan Weinsanto
Do you have access to a piezo drive? With this you could automate the position and removal of the puff pipette and this might help your issues with tip clogging and fear of drug application. I've used PSI of about 10-12 for pressure ejection for ~250 msec from a standard patch pipette (~5 mohm resistance) successfully. It's inevitable that clogging will happen but filtering and keeping all of your pipette gear clear may help minimize the frequency?
Unfortunately, I've never combined the method with calcium imaging so this is all the insight I have =(
Viewing the pressure wave is a basic way you confirm puff application so it doesn't seem to be a very favorable method of administration; but it might be your best/only option.
Dear Ivan,
I agree with most of the comments above. Indeed it is not an ideal set up to puff apply drugs onto the cells without activating mechanosensitive channels.I think you using a larger tip electrode is a good idea but it will also increase the leak of contents from the tip of the electrode. You may want to fill the very tip with extracellular fluid so that it does not cause any desensitisation due to premature leakage of the drug from the tip. In this way, you can get close to the cell as much as possible - and almost touch the soma and then apply minimal pressure or just wait for the drug to leak onto the cell with intervals. Also it may be a good idea to use multiple tip electrodes in case one blocks others may be still open and allow the application. Another option could be to use a U-Tube that puffs the drug only when the flow is stopped. I have used U-tube in isolated cells but never tried it in brain slices. It may be ok in very superficial cells.
Best wishes, Refik
- Badges
- Science topic
- Similar topics
- Papers