Immediately after brain isolation hippocampus was isolated and with the help of tissue chopper 300 micron thick slices were prepared. The slices were incubated at 32 c in ACSF for about 60 minutes.When we tried to record the data with MEA2100 system I did not find any spontaneous activity with the slice.
Dear Medapati,
First of all, you may need to check your cells under a microscope and if they look OK and alive. Secondly you may need to check individual cells with an extracellular electrode filled with extracellular solution to see if they have spontaneous activity. Once you confident that you have viable neurons and they are spontaneously active than you can check with multi electrodes. Otherwise you may be wasting your time. If your cells are spontaneously active than you may depolarise them by slightly increasing your extracellular K concentration gradually, say from normally 3 mM to 5 or 10 mM should give you spontaneous activity. For technical 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...
Technical Report Research Methods and Technical Considerations: Answers to ov...
Rajesh,
I believe that the Kanjhan's solution is the best in this case
Good look
I agree with what Refik stated, but would also add the following remark:
- adequate contact between the MEA and slice is hard to attain and vitally important!
this is especially hard with unperforated MEAs (like the MEA2100 I think)
Myself and Rajesh Medapati are trying to validate the procedures for slice electrophysiology in our laboratory.
We have a primary question that, how can we record the neuronal activity using extracellular electrodes as we have MEA2100 -64 electrode recording system which is not provided with extracellular electrodes for checking the viability.
We are using McLwain tissue chopper for making the hippocampal slices and MEA suggested to use vibrotome. Will there be a great difference between using vibrotome and tissue chopper for viability of slice.
Further, how can we check the viability of the cells using microscope by looking at the cells.
A vibratome will do a better job of preserving cells near the surface of the slice. With a tissue chopper, the first healthy cells you encounter will be deeper in the slice -- I could imagine this might be important for MEAs, but I have no experience with them.
Regarding looking at the slices, you can indeed make reasonable qualitative assessments by visual inspection (the above-mentioned extracellular electrode is a better test), but it's a little hard to describe how, exactly. For instance, in CA1 you want good contrast between the cell body layer, and the adjacent layers, and you want that boundary to be reasonably sharp and smooth. Precisely what constitutes "good contrast" and "sharp" boundaries is a little qualitative. If you could persuade a colleague with brain slice experience to take a look at your slices, they would probably be able to help you.
We use MEA2100 and compared chopped and vibratome cut slices. Vibratome cut is healthier. We put weight on the slice (silver coil, platinum would be better for sure) when we do the MEA recording on non-perforatted MEA.
Dear Medapati! Please listen to the advice of Refik, Brian and other colleagues who want to help you. You will learn the functional activity of neurons. For example, it would be disastrously low. You need to examine the reasons. I recommend you pay attention to the composition of the artificial CSF. For example, compare with our staff artificial cerebrospinal fluid. We did not have the problems that you have. Please, open the two papers in the attachment.
Vladimir
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Histological Techniques