I have come across a lot of work where investigators have patched dissociated or cultured intracardiac neurons, but have yet to see any papers where patching is done in situ. By in situ I mean an isolated in vitro preparation of intact ganglia. Also seen plenty of intracellular microelectrode recordings but this is not what I'm interested in. Thanks for your help in advance.
Hi Jesse,
Sounds like you are on a mission, a pretty hard one. I worked with intracardiac ganglion neurons, freshly isolated and recorded with perforated-patch clamp. I have also done sharp electrode recordings from pelvic ganglion neurons in situ. However, I have tried to patch in situ but gave up for the reasons you have listed above. I think the enzymes may work, but II would suggest that you remove the top 50 to 100 micron of the tissue with a vibratome. Then See if you can patch-clamp the soma on the surface, though you may still need to apply pressure to clear. I suggest you use a seperate electrode filled with extra cellular solution and use plenty of pressure to clear the soma.
Good luck and best wishes,
Refik
This has been done for chick ciliary ganglion neurons by Yawo. He sliced through the ganglion and patched the neurons. I've worked a lot on these cells and was astonished that he could access the cell membrane - for the reasons you give above. I met him at a meeting and he told me that he applied a collagenase through a pipette to expose the cell surface. Obviously this could cause untoward effects like removing glial cell contact or synaptic inputs but it might be a last resort method. One other method to explore is the removal of overlying material using a laser. I don't know how effective this is in animal ganglia but it was touted at one time for access to plant cells and would avoid the use of enzymes.
But maybe patch clamp is the wrong way to go here. Have you looked into 'discontinuous single electrode' voltage clamp? This method used a single sharp electrode and clamps by rapidly switching from current to voltage clamp. There are issues to deal with - in particular a limited control of larger currents - but the advantage is that you do not have to disturb the cell environment, as would be inevitable for patching.
Here is a recent paper using this method:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626565/
Good luck and have fun exploring - if you can pull this off you could make a considerable technical contribution :)
ee
Thank you for your valuable input Refik and Elise. I will persevere for the moment and explore the options you have suggested.
I can finally tell you we did it!!!! Thank you so much Elise and Refik for your help all those years ago. The Yawo reference was pivotal. Check out our new paper: 10.1152/ajpheart.00020.2020
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