We are performing patch clamp on motoneurons in ventral horn of neonatal rabbit spinal cord. The problem is very low cell content in slices of SC (one good cell per 2-3 slices). We tried to dissect SC segment from ventral or dorsal site of vertebral collumn but found no difference. We use vertical vibratome VF-300. Segment of lumbar spinal cord embedded in low melting agarose and cut in to 300mkm slices in ice cold (slash) ACSF. We tried to use single HEPES ACSF or 3 solutions: one for slicing (NaCl substituted with sucrose or NMDG), one for incubation (low Ca high Mg), one for recording (normal Ca/Mg concentrations) with HEPES. All solution according to publications with good results. We also check slicing conditions with hippocampus and get excellent slices with high cell content and good electrophysiological properties of neurons. We also tried several times to use 5-10 days old rat spinal cord slicing and didn’t get cells in slices at all. There is many papers using patch clamp in SC slices of rats with good result. Is any one can help to understand what is the secret?
Hello Ivan
This is a fantastic question. I regularly perform whole-cell recordings in adult mouse spinal cord preparations, from spinal cord injured mice no less. Although we use slightly different solutions in my lab, you are doing all of the right things: low Ca/High Mg, using a cold sucrose ACSF cutting solution etc. In my lab we do a transcardiac perfusion with the cold cutting solution before dissecting out the spinal cord. We do not embedd the cord in agar and instead we mount the cord on a agar block (just to stabilise). I have also found that the speed of dissection is critical, the faster the better. The details of my procedures can be found here:
Article Functional changes in deep dorsal horn interneurons followin...
I am happy for you to contact me and we can skype if you'd like more detail.
You should be additionally aware that, while recording from spinal cord slice preparations is common, recording from motoneurons in slice preparations is notoriously difficult. Much of the published spinal cord slice work is done is in superficial dorsal horn or, less commonly, in deep dorsal horn. There are a few brave souls who have successfully, and regularly, recorded from ventral horn motoneurons. Below are a few that I know, but I'm certain there are more out there.
Rob Brownstone and his group published a very detailed paper a few years ago on how he prepared the specimen:
Article An in vitro spinal cord slice preparation for recording from...
Ron Harris-Warrickand his then post-doc Andrea Husch have also had some success in this domain:
Article Spinal Cord Injury Induces Serotonin Supersensitivity withou...
Lastly, Simon Gosnach's group a few years ago developed a novel technique that involves cutting a notch in the dorsal laminae to reveal ventral interneuron populations and successfully record from these populations:
Article Whole Cell Recordings From Visualized Neurons in the Inner L...
Happy patching!
Hi Michelle. Thank you very much for fast answer. I will be happy to skipe with you. What is best time to call? As per my understanding you mean that to get good cell content in ventral horn is more difficult that in dorsal. How many patchable looking cells in your preparation? As per your suggestion we tried today not to embedded cord in to agarose but keep it in to groove on agar block just glue the distal tip of segment ( we use vertical slicer). The cutting was not successful because cord was moved. We didn’t glue cord along to block assuming that it will be killed by toxicity. Do you really glue cord to agar block all way along? Is it ok to do? Also interesting why you dissect cord from ventral site. We did both (ventral and dorsal dissection and found that much ease to do it from dorsal site) . We also don’t remove dura mater because in neonatal rabbits its perfectly cuts through it.
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