Hi all,
Do you know of any strategy to increase AMPAR mEPSCs frequency for cultured hippocampal neurons? Methods on the side of culturing and/or of recording environment are much appreciated!
Thank you so much!
Some background: Our lab makes cultures from embryonic hippocampi at E18 - E19 and co-cultures them with postnatal astrocytes (Banker method) in neurobasal + B27 + glutamax media. At DIV 7, to prevent over-proliferation of glial cells, we treat them with 2 uM of AraC for 12 - 24 h. This method works really well to keep the neurons healthy and suitable for transfection, immunostaining, and some imaging application. However, the neurons seem to not have robust and consistent spontaneous mini and/or evoked EPSCs, sometimes even up to DIV 19. When saying not robust, I meant they were actually super quiet, or just have 1- 2 events every minute. If you have any suggestion, please let me know!
Currently, I am trying to compare between different B27 supplement lots and N2 supplement. I am reluctant to change anything too dramatically since I already collected more than half of the data for this project using this culture system.
In term of recording, I have tried increasing the KCl concentration in the extracellular environment up to 8.5 mM (from a baseline of 5.4 mM) but didn't see much increase in the frequency. Intsead, the holding current just dropped really terribly (from - 60 pA to - 300 or 400 pA). The extracellular solution I use is mimicking the recipe of Hibernate E (created by Brain Bit), as recommended by Jonathan Ting in this post below. This solution keeps the neurons really healthy up to 3- 4 hours outside of the media. And I can record up to 1 h long in whole-cell mode from each neurons.
https://www.researchgate.net/post/Patch_clamping_cultured_neurons-can_anyone_help
Best,
Huong
Dear Huong, have you quantified the density of axon terminals on the neurons, with FM1- 43 or by IF? If density is too low, all the tricks to increase spontaneous release will fail. With FM1-43, you should see almost continous surface staining after rinsing. If there are large gaps, then you cell density might be a bit low. I hope this helps
Hi Huong,
What is the density of your culture? How did you change the medium?
Actually, I came up to the similar problem a while ago, although I was using rat cortical cultures. The problem for me then was that, evoked events were robust but miniature events were very little. My culture condition is a little different from yours. please find the detail in my previous poster following,
https://www.researchgate.net/post/Why_rat_cortical_culture_has_low_mEPSCs_freqency_but_high_sEPSCs_frequency_at_14DIV
Long story short. Eventually, I plated neurons at the density ~2.5*10^5/mL and adopted a medium changing method as following, adding ~200 ul fresh medium every other day in the first week and replacing 1/2 of the medium after. mEPSCs and mIPSCs were recorded around 21DIV. Roughly the frequency for both is reasonable, about 0.5 Hz, although there is variation among different cultures.
Also, is there any way for you to check the morphology of the neurons? I routinely did GFP, along with others, transfection at 8DIV. My impression is that, for culture with high frequency events I can see dense mature spines, i.e. mushroom shape, however, the spins more looked like filopodia in culture with low mini frequency.
I thought, and still think so, maybe there are several steps for synapse development in culture and somehow neurons could be fixed at certain step. It is low eEPSP and mEPSC for you and high eEPSC and low mEPSC in my case.
Hope this can help.
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